Functional influenza virus-like particles (VLPs)

ABSTRACT

The present invention discloses and claims virus like particles (VLPs) that express and/or contains seasonal influenza virus proteins, avian influenza virus proteins and/or influenza virus proteins from viruses with pandemic potential. The invention includes vector constructs comprising said proteins, cells comprising said constructs, formulations and vaccines comprising VLPs of the inventions. The invention also includes methods of making and administrating VLPs to vertebrates, including methods of inducing substantial immunity to either seasonal and avian influenza, or at least one symptom thereof.

This application is a divisional of U.S. patent application Ser. No.11/582,540, filed Oct. 18, 2006, now U.S. Pat. No. 8,080,255, which is acontinuation in part of U.S. application Ser. No. 10/617,569, filed Jul.11, 2003, each of which is incorporated herein by reference in itsentirety for all purposes. now U.S. Pat. No. 8,592,197 This applicationalso claims priority to U.S. provisional application Ser. No.60/727,513, filed Oct. 18, 2005, U.S. provisional application60/780,847, filed Mar. 10, 2006, U.S. provisional application Ser. No.60/800,006, filed May 15, 2006, U.S. provisional application Ser. No.60/831,196, filed Jul. 17, 2006, U.S. provisional application Ser. No.60/832,116, filed Jul. 21, 2006, and U.S. provisional application Ser.No. 60/845,495, filed Sep. 19, 2006, all of which are incorporatedherein by reference in their entireties for all proposes.

The contents of the following electronically-submitted text file isincorporated by reference herein in its entirety: A computer readableformat copy of the Sequence Listing (filename:NOVV_(—)002_(—)07US_SeqList.txt, date recorded: Nov. 15, 2011, file size119 kb).

BACKGROUND OF INVENTION

Influenza virus is a member of Orthomyxoviridae family (for review, seeMurphy and Webster, 1996). There are three subtypes of influenza virusesdesignated A, B, and C. The influenza virion contains a segmentednegative-sense RNA genome. The influenza virion includes the followingproteins: hemagglutinin (HA), neuraminidase (NA), matrix (M1), protonion-channel protein (M2), nucleoprotein (NP), polymerase basic protein 1(PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA),and nonstructural protein 2 (NS2) proteins. The HA, NA, M1, and M2 aremembrane associated, whereas NP, PB1, PB2, PA, and NS2 are nucleocapsidassociated proteins. The NS1 is the only nonstructural protein notassociated with virion particles but specific for influenza-infectedcells. The M1 protein is the most abundant protein in influenzaparticles. The HA and NA proteins are envelope glycoproteins,responsible for virus attachment and penetration of the viral particlesinto the cell, and the sources of the major immunodominant epitopes forvirus neutralization and protective immunity. Both HA and NA proteinsare considered the most important components for prophylactic influenzavaccines.

Influenza virus infection is initiated by the attachment of the virionsurface HA protein to a sialic acid-containing cellular receptor(glycoproteins and glycolipids). The NA protein mediates processing ofthe sialic acid receptor, and virus penetration into the cell depends onHA-dependent receptor-mediated endocytosis. In the acidic confines ofinternalized endosomes containing an influenza virion, the HA proteinundergoes conformational changes that lead to fusion of viral and hostcell membranes followed by virus uncoating and M2-mediated release of M1proteins from nucleocapsid-associated ribonucleoproteins (RNPs), whichmigrate into the cell nucleus for viral RNA synthesis. Antibodies to HAmolecule can prevent virus infection by neutralizing virus infectivity,whereas antibodies to NA proteins mediate their effect on the earlysteps of viral replication.

Inactivated influenza A and B virus vaccines are licensed currently astrivalent vaccines for parenteral administration. These trivalentvaccines are produced as monovalent bulk in the allantoic cavity ofembryonated chick eggs, purified by rate zonal centrifugation or columnchromatography, inactivated with formalin or β-propiolactone, andformulated as a blend of the two strains of type A and the type B strainof influenza viruses in circulation among the human population for agiven year. The available commercial influenza vaccines are whole virus(WV) or subvirion (SV; split or purified surface antigen) virusvaccines. The WV vaccine contains intact, inactivated virions. SVvaccines treated with solvents such as tri-n-butyl phosphate(Flu-Shield, Wyeth-Lederle) contain nearly all of the viral structuralproteins and some of the viral envelopes. SV vaccines solubilized withTriton X-100 (Fluzone, Sanofi-Aventis; Fluvirin, Novartis) containaggregates of HA monomers, NA, and NP principally, although residualamounts of other viral structural proteins are present. A liveattenuated cold-adapted virus vaccine (FluMist, Medlmmune) was grantedmarketing approval recently by the FDA for commercial usage as anintranasally delivered vaccine indicated for active immunization and theprevention of disease caused by influenza A and B viruses in healthychildren and adolescents, 5-17 years of age and healthy adults 18-49years of age.

Several recombinant products have been developed as recombinantinfluenza vaccine candidates. These approaches have focused on theexpression, production, and purification of influenza virus type A HAand NA proteins, including expression of these proteins usingbaculovirus infected insect cells (Crawford et al, 1999; Johansson,1999; Treanor et al., 1996), viral vectors (Pushko et al., 1997;Berglund et al., 1999), and DNA vaccine constructs (Olsen et al., 1997).

Crawford et al. (1999) demonstrated that influenza HA expressed inbaculovirus infected insect cells is capable of preventing lethalinfluenza disease caused by avian H5 and H7 influenza subtypes. At thesame time, another group demonstrated that baculovirus-expressedinfluenza HA and NA proteins induce immune responses in animals superiorto those induced by a conventional vaccine (Johansson et al., 1999).Immunogenicity and efficacy of baculovirus-expressed hemagglutinin ofequine influenza virus was compared to a homologous DNA vaccinecandidate (Olsen et al., 1997). Taken together, the data demonstratedthat a high degree of protection against influenza virus challenge canbe induced with recombinant HA or NA proteins, using variousexperimental approaches and in different animal models.

Lakey et al. (1996) showed that a baculovirus-derived influenza HAvaccine was well-tolerated and immunogenic in human volunteers in aPhase I dose escalation safety study. However, results from Phase IIstudies conducted at several clinical sites in human volunteersvaccinated with several doses of influenza vaccines comprised of HAand/or NA proteins indicated that the recombinant subunit proteinvaccines did not elicit protective immunity [G. Smith, Protein Sciences;M. Perdue, USDA, Personal Communications]. These results indicated thatconformational epitopes displayed on the surface of HA and NA peplomersof infectious virions were important in the elicitation of neutralizingantibodies and protective immunity.

Regarding the inclusion of other influenza proteins in recombinantinfluenza vaccine candidates, a number of studies have been carried out,including the experiments involving influenza nucleoprotein, NP, aloneor in combination with M1 protein (Ulmer et al., 1993; Ulmer et al.,1998; Zhou et al., 1995; Tsui et al., 1998). These vaccine candidates,which were composed of quasi-invariant inner virion proteins, elicited abroad spectrum immunity that was primarily cellular (both CD4⁺ and CD8⁺memory T cells). These experiments involved the use of the DNA or viralgenetic vectors. Relatively large amounts of injected DNA were needed,as results from experiments with lower doses of DNA indicated little orno protection (Chen et al., 1998). Hence, further preclinical andclinical research may be required to evaluate whether such DNA-basedapproaches involving influenza NP and M1 are safe, effective, andpersistent.

Recently, in an attempt to develop more effective vaccines forinfluenza, particulate proteins were used as carriers of influenza M2protein epitopes. The rationale for development of an M2-based vaccinewas that in animal studies protective immunity against influenza waselicited by M2 proteins (Slepushkin et al., 1995). Neirynck et al.(1999) used a 23-aa long M2 transmembrane domain as an amino terminalfusion partner with the hepatitis B virus core antigen (HBcAg) to exposethe M2 epitope(s) on the surface of HBcAg capsid-like particles.However, in spite of the fact that both full-length M2 protein andM2-HBcAg VLP induced detectable antibodies and protection in mice, itwas unlikely that future influenza vaccines would be based exclusivelyon the M2 protein, as the M2 protein was present at low copy number pervirion, was weakly antigenic, was unable to elicit antibodies that boundfree influenza virions, and was unable to block virus attachment to cellreceptors (i.e. virus neutralization).

Since previous research has shown that the surface influenzaglycoproteins, HA and NA, are the primary targets for elicitation ofprotective immunity against influenza virus and that M1 provides aconserved target for cellular immunity to influenza, a new vaccinecandidate may include these viral antigens as a protein macromolecularparticle, such as virus-like particles (VLPs). Further, the particlewith these influenza antigens may display conformational epitopes thatelicit neutralizing antibodies to multiple strains of influenza viruses.

Several studies have demonstrated that recombinant influenza proteinscould self-assemble into VLPs in cell culture using mammalian expressionplasmids or baculovirus vectors (Gomez-Puertas et al., 1999; Neumann etal., 2000; Latham and Galarza, 2001). Gomez-Puertas et al. (1999)demonstrated that efficient formation of influenza VLP depends on theexpression levels of viral proteins. Neumann et al. (2000) established amammalian expression plasmid-based system for generating infectiousinfluenza virus-like particles entirely from cloned cDNAs. Latham andGalarza (2001) reported the formation of influenza VLPs in insect cellsinfected with recombinant baculovirus co-expressing HA, NA, M1, and M2genes. These studies demonstrated that influenza virion proteins mayself-assemble upon co-expression in eukaryotic cells.

SUMMARY OF INVENTION

The present invention provides for a vaccine comprising an influenzaVLP, wherein said VLP comprises influenza M1, HA and NA proteins,wherein said vaccine induces substantial immunity to influenza virusinfection in an animal susceptible to influenza. In one embodiment, saidM1 protein is derived from a different influenza virus strain ascompared to the HA and NA proteins. In another embodiment, said HAand/or NA exhibit hemagglutinin activity and/or neuraminidase activity,respectfully. In another embodiment, said influenza VLP comprisesseasonal influenza virus HA and NA proteins. In another embodiment, saidinfluenza VLP comprises avian influenza HA and NA proteins.

The present invention also provides for a method of inducing substantialimmunity to influenza virus infection in an animal susceptible toinfluenza, comprising administering at least one effective dose of thevaccine comprising an influenza VLP. In one embodiment, said methodcomprises administering to an animal said influenza VLP orally,intradermally, intranasally, intramusclarly, intraperitoneally,intravenously, or subcutaneously.

The present invention also provides for a method of formulating avaccine that induces substantial immunity to influenza virus infectionto an animal susceptible to influenza, comprising adding to saidformulation an effective dose of an influenza VLP, wherein said VLPcomprises influenza M1, HA and NA proteins, wherein said vaccine inducessubstantial immunity to influenza virus infection to said animal. In oneembodiment, said VLP consists essentially of influenza M1, HA and NAproteins. In another embodiment, said VLP consists of influenza M1, HAand NA proteins.

The present invention also provides for a virus like particle (VLP)comprising an influenza virus M1 protein and influenza virus H5 and N1hemagglutinin and neuraminidase proteins. In one embodiment said M1protein is derived from a different influenza virus strain as comparedto the H5 and N1 proteins. In one embodiment, said H5 or N1 are from aH5N1 clade 1 influenza virus. In another embodiment, said H5 and N1 arefrom a H5N1 clade 2 influenza virus.

The invention also provides a macromolecular protein structurecontaining (a) a first influenza virus M1 protein and (b) an additionalstructural protein, which may include a second or more influenza virusM1 protein; a first, second or more influenza virus HA protein; a first,second, or more influenza virus NA protein; and a first, second, or moreinfluenza virus M2 protein. If the additional structural protein is notfrom a second or more influenza virus M1 protein, then both or allmembers of the group, e.g., first and second influenza M2 virus proteinsare included. As such, there is provided a functional influenza proteinstructure, including a subviral particle, VLP, or capsomer structure, ora portion thereof, a vaccine, a multivalent vaccine, and mixturesthereof consisting essentially of influenza virus structural proteinsproduced by the method of the invention. In a particularly preferredembodiment, the influenza macromolecular protein structure includesinfluenza virus HA, NA, and M1 proteins that are the expression productsof influenza virus genes cloned as synthetic fragments from a wild typevirus.

The macromolecular protein structure may also include an additionalstructural protein, for example, a nucleoprotein (NP), membrane proteinsfrom species other than noninfluenza viruses and a membrane protein froma non-influenza source, which are derived from avian or mammalianorigins and different subtypes of influenza virus, including subtype Aand B influenza viruses. The invention may include a chimericmacromolecular protein structure, which includes a portion of at leastone protein having a moiety not produced by influenza virus.

Prevention of influenza may be accomplished by providing amacromolecular protein structure that may be self-assembled in a hostcell from a recombinant construct. The macromolecular protein structureof the invention has the ability to self-assemble into homotypic orheterotypic virus-like particles (VLPs) that display conformationalepitopes on HA and NA proteins, which elicit neutralizing antibodiesthat are protective. The composition may be a vaccine composition, whichalso contains a carrier or diluent and/or an adjuvant. The functionalinfluenza VLPs elicit neutralizing antibodies against one or morestrains or types of influenza virus depending on whether the functionalinfluenza VLPs contain HA and/or NA proteins from one or more viralstrains or types. The vaccine may include influenza virus proteins thatare wild type influenza virus proteins. Preferably, the structuralproteins containing the influenza VLP, or a portion of thereof, may bederived from the various strains of wild type influenza viruses. Theinfluenza vaccines may be administered to humans or animals to elicitprotective immunity against one or more strains or types of influenzavirus.

The macromolecular protein structures of the invention may exhibithemagglutinin activity and/or neuraminidase activity.

The invention provides a method for producing a VLP derived frominfluenza by constructing a recombinant construct that encodes influenzastructural genes, including M1, HA, and at least one structural proteinderived from influenza virus. A recombinant construct is used totransfect, infect, or transform a suitable host cell with therecombinant baculovirus. The host cell is cultured under conditionswhich permit the expression of M1, HA and at least one structuralprotein derived from influenza virus and the VLP is formed in the hostcell. The infected cell media containing a functional influenza VLP isharvested and the VLP is purified. The invention also features anadditional step of co-transfecting, co-infecting or co-transforming thehost cell with a second recombinant construct which encodes a secondinfluenza protein, thereby incorporating the second influenza proteinwithin the VLP. Such structural proteins may be derived from influenzavirus, including NA, M2, and NP, and at least one structural protein isderived from avian or mammalian origins. The structural protein may be asubtype A and B influenza viruses. According to the invention, the hostcell may be a eukaryotic cell. In addition, the VLP may be a chimericVLP.

The invention also features a method of formulating a drug substancecontaining an influenza VLP by introducing recombinant constructsencoding influenza viral genes into host cells and allowingself-assembly of the recombinant influenza viral proteins into afunctional homotypic or heterotypic VLP in cells. The influenza VLP isisolated and purified and a drug substance is formulated containing theinfluenza VLP. The drug substance may further include an adjuvant. Inaddition, the invention provides a method for formulating a drugproduct, by mixing such a drug substance containing an influenza VLPwith a lipid vesicle, i.e., a non-ionic lipid vesicle. Thus, functionalhomotypic or heterotypic VLPs may bud as enveloped particles from theinfected cells. The budded influenza VLPs may be isolated and purifiedby ultracentrifugation or column chromatography as drug substances andformulated alone or with adjuvants such as Novasomes®, a product ofNovavax, Inc., as drug products such as vaccines. Novasomes®, whichprovide an enhanced immunological effect, are further described in U.S.Pat. No. 4,911,928, which is incorporated herein by reference.

The invention provides a method for detecting humoral immunity toinfluenza virus infection in a vertebrate by providing a test reagentincluding an effective antibody-detecting amount of influenza virusprotein having at least one conformational epitope of an influenza virusmacromolecular structure. The test reagent is contacted with a sample ofbodily fluid from a vertebrate to be examined for influenza virusinfection. Influenza virus specific antibodies contained in the sampleare allowed to bind to the conformational epitope of an influenza virusmacromolecular structure to form antigen-antibody complexes. Thecomplexes are separated from unbound complexes and contacted with adetectably labeled immunoglobulin-binding agent. The amount of thedetectably labeled immunoglobulin-binding agent that is bound to thecomplexes is determined.

Influenza virus may be detected in a specimen from an animal or humansuspected of being infected with the virus by providing antibodies,which have a detectable signal producing label, or are attached to adetectably labeled reagent, having specificity to at least oneconformational epitope of the particle of the influenza virus. Thespecimen is contacted with antibodies and the antibodies are allowed tobind to the influenza virus. The presence of influenza virus in thespecimen is determined by means of the detectable label.

The invention provides methods for treatment, prevention, and generatinga protective immune response by administering to a vertebrate aneffective amount of the composition of the invention.

Alternatively, the influenza VLP drug substance may be formulated aslaboratory reagents used for influenza virus structure studies andclinical diagnostic assays. The invention also provides a kit fortreating influenza virus by administering an effective amount of acomposition of the invention and directions for use.

The invention also provides for a VLP comprising HA, NA and M1 proteinsderived from an avian influenza virus which can cause morbidity ormortality in a vertebrate. In one embodiment, said HA, NA and M1proteins are derived from an avian influenza type A virus. In anotherembodiment the HA is selected from the group consisting of H1, H2, H3,H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16 and the NAis selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8and N9. In a further embodiment, said HA and NA proteins are H5 and N1,respectively. In another embodiment, said HA and NA proteins are H9 andN2, respectively. In another embodiment, said HA and/or NA exhibitshemagglutinin activity and/or neuraminidase activity, respectfully. Inone embodiment, the VLP consists essentially of HA, NA and M1 proteins,i.e., these are substantially the only influenza proteins in the VLP.

The invention also provides for a method of producing a VLP, comprisingtransfecting vectors encoding avian influenza virus proteins into asuitable host cell and expressing said avian influenza virus proteinsunder condition that allow VLPs to be formed. In one embodiment, thismethod involves transfecting a host cell with recombinant DNA moleculesthat encode only the HA, NA and M1 influenza proteins.

The invention also comprises an antigenic formulation comprising a VLPcomprising HA, NA and M1 proteins derived from an avian influenza viruswhich can cause morbidity or mortality in a vertebrate. In anotherembodiment, the HA is selected from the group consisting of H1, H2, H3,H4, H5, H6, H7, H8, H9, H10, H11, H112, H13, H14, H15 and H16 and the NAis selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8and N9. In a further embodiment, said HA and NA proteins are H5 and N1,respectively. In another embodiment, said HA and NA proteins are H9 andN2, respectively. In a further embodiment, said antigenic formulation isadministered to the subject orally, intradermally, intranasally,intramusclarly, intraperitoneally, intravenously, or subcutaneously.

The invention further provides for a method of vaccinating a vertebrateagainst avian influenza virus comprising administering to saidvertebrate a protection-inducing amount of a VLP comprising HA, NA andM1 proteins derived from an avian influenza virus.

This invention also comprises a method of inducing substantial immunityto influenza virus infection or at least one symptom thereof in asubject, comprising administering at least one effective dose of aninfluenza VLP. In one embodiment, said VLP consists essentially of HA,NA and M1. In another embodiment, said VLP comprises influenza proteins,wherein said influenza proteins consist of HA, NA and M1. In anotherembodiment, said HA and/or NA exhibits hemagglutinin activity and/orneuraminidase activity, respectfully.

This invention also comprises a method of inducing substantial immunityto influenza virus infection or at least one symptom thereof in asubject, comprising administering at least one effective dose of anavian influenza VLP. In one embodiment, said influenza VLP consistsessentially of avian HA, NA and M1. In another embodiment, saidinfluenza VLP comprises influenza proteins, wherein said influenzaproteins consist of avian HA, NA and M1

This invention further comprises a method of inducing substantialimmunity to influenza virus infection or at least one symptom thereof ina subject, comprising administering at least one effective dose of aseasonal influenza VLP. In one embodiment, said influenza VLP consistsessentially of seasonal HA, NA and M1. In another embodiment, saidinfluenza VLP comprises influenza proteins, wherein said influenzaproteins consist of seasonal HA, NA and M1.

This invention further comprises a method of inducing substantialimmunity to influenza virus infection or at least one symptom thereof ina subject, comprising administering at least one effective dose of atleast one seasonal influenza VLP. In one embodiment, said influenza VLPcomprises seasonal influenza HA, NA and M1. In another embodiment, saidinfluenza VLP consists essentially of seasonal influenza HA, NA and M1.

This invention further comprises a method of inducing a substantiallyprotective antibody response to influenza virus infection or at leastone symptom thereof in a subject, comprising administering at least oneeffective dose of an influenza VLP.

This invention comprises a method of inducing a substantially protectivecellular immune response to influenza virus infection or at least onesymptom thereof in a subject, comprising administering at least oneeffective dose of an influenza VLP.

This invention further comprises a method of formulating a vaccine thatinduces substantial immunity to influenza virus infection or at leastone symptom thereof to a subject, comprising adding to said formulationan effective dose of an influenza VLP. In one embodiment, saidsubstantial immunity to influenza virus infection or at least onesymptom thereof is delivered in one dose. In another embodiment, saidsubstantial immunity to influenza virus infection or at least onesymptom thereof is delivered in multiple doses.

This invention further comprises a vaccine comprising an influenza VLP,wherein said vaccine induces substantial immunity to influenza virusinfection or at least one symptom thereof when administered to asubject. In one embodiment, said influenza VLP is an avian influenzaVLP. In another embodiment, said influenza VLP is a seasonal influenzaVLP.

This invention further comprises an antigenic formulation comprising aninfluenza VLP, wherein said vaccine induces substantial immunity toinfluenza virus infection or at least one symptom thereof whenadministered to a subject. In one embodiment, said influenza VLP is anavian influenza VLP. In another embodiment, said influenza VLP is aseasonal influenza VLP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence of avian influenza A/HongKong/1073/99 (H9N2) virus neuraminidase (NA) gene (SEQ ID NO:1).

FIG. 2 depicts the nucleotide sequence of avian influenza A/HongKong/1073/99 (H9N2) virus hemagglutinin (HA) gene (SEQ ID NO:2).

FIG. 3 depicts the nucleotide sequence of avian influenza A/HongKong/1073/99 (H9N2) virus matrix protein M1 (M1) gene (SEQ ID NO:3).

FIG. 4 depicts the transfer vectors for construction of recombinantbaculoviruses for expression of avian influenza A/Hong Kong/1073/99(H9N2) HA, NA, and M1 proteins. FIG. 4A depicts a transfer vector forexpression of individual genes and FIG. 4B depicts the transfer vectorfor multi-expression of the genes.

FIG. 5 depicts the expression of avian influenza A/Hong Kong/1073/99(H9N2) virus HA, NA, and M1 proteins in Sf-9S cells.

FIG. 6 depicts the purification of avian influenza A/Hong Kong/1073/99(H9N2) VLPs by the sucrose density gradient method.

FIG. 7 depicts the detection of influenza virus protein by gelfiltration chromatography. The antibodies used in the Western blotanalyses are as follows: (A) rabbit anti-H9N2; (b) murine anti-M1 mAb;and (C) murine anti-BACgp64.

FIG. 8 depicts the detection of avian influenza A/Hong Kong/1073/99(H9N2) proteins including subviral particles, VLP, and VLP complexes, byelectron microscopy.

FIG. 9 depicts the hemagglutination activity of purified avian influenzaA/Hong Kong/1073/99 (H9N2) VLPs.

FIG. 10 depicts the neuraminidase activity of purified avian influenzaA/Hong Kong/1073/99 (H9N2) VLPs.

FIG. 11 depicts the immunization and bleed schedule for theimmunogenicity study of recombinant influenza with purified avianinfluenza A/Hong Kong/1073/99 (H9N2) VLPs in mice.

FIG. 12 depicts the results of an immunogenicity study in mice immunizedwith recombinant influenza H9N2 VLPs. FIG. 12A depicts sera from BALB/cmice immunized with recombinant VLPs comprised of HA, NA, and M1proteins from avian influenza virus type A/H9N2/Hong Kong/1073/99. FIG.12B depicts sera from New Zealand white rabbits immunized withinactivated avian influenza virus type A H9N2 were reacted with Westernblots containing inactivated avian influenza virus type A H9N2 (lanes 1and 3) or cold-adapted avian influenza virus type A H9N2 (lanes 2 and4).

FIG. 13 depicts the geometric mean antibody responses in BALB/c miceafter a primary and secondary immunization.

FIG. 14 depicts serum hemagglutinin inhibition (H1) responses in BALB/cmice.

FIG. 15 depicts weight loss (%) in BALB/c mice challenged with H9N2influenza.

FIG. 16 depicts lung virus titers at 3 and 5 days post challenge withH9N2.

FIGS. 17A, 17B and 17C depict mice antibody response toA/Fujian/411/2002 when immunized with H3N2 VLP.

FIGS. 18 A and B depict mice IgG antibody isotypes

FIG. 19 hemagglutinin inhibition (HI) antibody responses in SD Ratsimmunized with H9N2 VLP vaccine.

FIGS. 20A and 20B depict hemagglutinin inhibition (HI) antibodyresponses to different doses of H9N2 VLPs with and without adjuvant inBALB/c mice.

FIG. 21 depicts serum hemagglutinin inhibition (HI) responses in BALB/cmice between different doses of VLPs.

FIG. 22 depicts serum hemagglutinin inhibition (HI) responses inferrets.

FIG. 23 depicts serum hemagglutinin inhibition (HI) responses from serumpulled on days 21 and 42 from ferrets after administration of differentstrains of H3N2 VLPs.

FIG. 24 depicts anti-HA Antibody (Endpoint Dilution Titer) of miceinoculated intramuscularly with H5N1 (Vietnam/1203/2003) VLPs at lowdoses.

FIG. 25 depicts anti-HA Antibody (Endpoint Dilution Titer) of miceinoculated intranasally with H5N1 (Vietnam/1203/2003) VLPs at low doses.

FIG. 26 depicts an example for manufacturing, isolating and purifyingVLPs of the invention.

FIG. 27 depicts mice inoculated with H3N2 VLPs given intramuscularly andsubsequently challenged intranasally with A/Aichi/2/68×31 (H3N2) virus.

FIG. 28 depicts mice inoculated with H3N2 VLPs given intranasally andsubsequently challenged intranasally with A/Aichi/2/68×31 (H3N2) virus.

FIG. 29 depicts virus shedding in nasal washes of ferret inoculated withH9N2 VLP vaccine and subsequently challenged intranasally with H9N2virus.

FIG. 30A, 30B, 30C, 30D, 30E, 30F, 30G, 30H depicts hemagglutinininhibition (HI) antibody responses in mice after inoculation withdifferent doses of A/Fujian/411/2002 (H3N2) VLPs intramuscularly orintranasally tested against different H3N2 strains of influenza viruses.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “baculovius,” also known as baculoviridae,refers to a family of enveloped DNA viruses of arthropods, members ofwhich may be used as expression vectors for producing recombinantproteins in insert cell cultures. The virion contains one or morerod-shaped nucleocapsids containing a molecule of circular supercoileddouble-stranded DNA (Mr 54×10⁶−154×10⁶). The virus used as a vector isgenerally Autographa californica nuclear polyhedrosis virus (NVP).Expression of introduced genes is under the control of the strongpromoter that normally regulates expression of the polyhedron proteincomponent of the large nuclear inclusion in which the viruses areembedded in the infected cells.

As used herein, the term “derived from” refers to the origin or source,and may include naturally occurring, recombinant, unpurified, orpurified molecules. The proteins and molecules of the present inventionmay be derived from influenza or non-influenza molecules.

As used herein the term “first” influenza virus protein, i.e., a firstinfluenza virus M1 protein, refers to a protein, such as M1, HA, NA, andM2, that is derived from a particular strain of influenza virus. Thestrain or type of the first influenza virus differs from the strain ortype of the second influenza virus protein. Thus, “second” influenzavirus protein, i.e., the second influenza virus M1 protein, refers to aprotein, such as M1, HA, NA, and M2, that is derived from a secondstrain of influenza virus, which is a different strain or type than thefirst influenza virus protein.

As used herein, the term “hemagglutinin activity” refers to the abilityof HA-containing proteins, VLPs, or portions thereof to bind andagglutinate red blood cells (erythrocytes).

As used herein, the term “neuraminidase activity” refers to theenzymatic activity of NA-containing proteins, VLPs, or portions thereofto cleave sialic acid residues from substrates including proteins suchas fetuin.

As used herein, the term “heterotypic” refers to one or more differenttypes or strains of virus.

As used herein, the term “homotypic” refers to one type or strain ofvirus.

As used herein, the term “macromolecular protein structure” refers tothe construction or arrangement of one or more proteins.

As used herein, the term “multivalent” vaccine refers to a vaccineagainst multiple types or strains of influenza virus.

As used herein, the term “non-influenza” refers to a protein or moleculethat is not derived from influenza virus.

As used herein, the term “vaccine” refers to a preparation of dead orweakened pathogens, or of derived antigenic determinants, that is usedto induce formation of antibodies or immunity against the pathogen. Avaccine is given to provide immunity to the disease, for example,influenza, which is caused by influenza viruses. The present inventionprovides vaccine compositions that are immunogenic and provideprotection. In addition, the term “vaccine” also refers to a suspensionor solution of an immunogen (e.g. VLP) that is administered to avertebrate to produce protective immunity, i.e., immunity that reducesthe severity of disease associated with infection.

As used herein the term “substantial immunity” refers to an immuneresponse in which when VLPs of the invention are administered to avertebrate there is an induction of the immune system in said vertebratewhich results in the prevention of influenza infection, amelioration ofinfluenza infection or reduction of at least one symptom related toinfluenza virus infection in said vertebrate. Substantial immunity mayalso refer to a hemagglutination inhibition (HI) titer of ≧40 in amammal wherein the VLPs of the invention have been administered and haveinduced an immune response.

As used herein the term “adjuvant” refers to a compound that, when usedin combination with a specific immunogen (e.g. a VLP) in a formulation,augments or otherwise alters or modifies the resultant immune response.Modification of the immune response includes intensification orbroadening the specificity of either or both antibody and cellularimmune responses. Modification of the immune response can also meandecreasing or suppressing certain antigen-specific immune responses.

As used herein the term “immune stimulator” refers to a compound thatenhances an immune response via the body's own chemical messengers(cytokines). These molecules comprise various cytokines, lymphokines andchemokines with immunostimulatory, immunopotentiating, andpro-inflammatory activities, such as interleukins (e.g., IL-1, IL-2,IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage(GM)-colony stimulating factor (CSF)); and other immunostimulatorymolecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1;B7.2, etc. The immune stimulator molecules can be administered in thesame formulation as the influenza VLPs, or can be administeredseparately. Either the protein or an expression vector encoding theprotein can be administered to produce an immunostimulatory effect.

As used herein an “effective dose” generally refers to that amount ofthe VLP of the invention sufficient to induce immunity, to preventand/or ameliorate influenza virus infection or to reduce at least onesymptom of influenza infection and/or to enhance the efficacy of anotherdose of a VLP. An effective dose may refer to the amount of the VLPsufficient to delay or minimize the onset of an influenza infection. Aneffective dose may also refer to the amount of the VLP that provides atherapeutic benefit in the treatment or management of influenzainfection. Further, an effective dose is the amount with respect to theVLPs of the invention alone, or in combination with other therapies,that provides a therapeutic benefit in the treatment or management of aninfluenza viral infection. An effective dose may also be the amountsufficient to enhance a subject's (e.g., a human's) own immune responseagainst a subsequent exposure to influenza virus. Levels of immunity canbe monitored, e.g., by measuring amounts of neutralizing secretoryand/or serum antibodies, e.g., by plaque neutralization, complementfixation, enzyme-linked immunosorbent, or microneutralization assay. Inthe case of a vaccine, an “effective dose” is one that prevents diseaseor reduces the severity of symptoms.

As used herein the term “avian influenza virus” refers to influenzaviruses found chiefly in birds but that can also infect humans or otheranimals. In some instances, avian influenza viruses may be transmittedor spread from one human to another. An avian influenza virus thatinfects humans has the potential to cause an influenza pandemic, i.e.,morbidity and/or mortality in humans. A pandemic occurs when a newstrain of influenza virus (a virus in which human have no naturalimmunity) emerges, spreading beyond individual localities, possiblyaround the globe, and infecting many humans at once.

As used herein the term “seasonal influenza virus” refers to theinfluenza viral strains that have been determined to be passing withinthe human population for a given influenza season based onepidemiological surveys conducted by National Influenza Centersworldwide. These epidemiological studies, and some isolated influenzaviruses, are sent to one of four World Health Organization (WHO)reference laboratories, one of which is at the Centers for DiseaseControl and Prevention (CDC) in Atlanta for detailed testing. Theselaboratories test how well antibodies made to the current vaccine reactto the circulating virus and new flu viruses. This information, alongwith information about flu activity, is summarized and presented to anadvisory committee of the U.S. Food and Drug Administration (FDA) and ata WHO meeting. These meetings result in the selection of three viruses(two subtypes of influenza A viruses and one influenza B virus) to gointo flu vaccines for the following fall and winter. The selectionoccurs in February for the northern hemisphere and in September for thesouthern hemisphere. Usually, one or two of the three virus strains inthe vaccine changes each year.

As used herein the term “substantially protective antibody response”refers to an immune response mediated by antibodies against an influenzavirus, which is exhibited by a vertebrate (e.g., a human), that preventsor ameliorates influenza infection or reduces at least one symptomthereof. VLPs of the invention can stimulate the production ofantibodies that, for example, neutralizing antibodies that blockinfluenza viruses from entering cells, blocks replication of saidinfluenza virus by binding to the virus, and/or protect host cells frominfection and destruction.

As used herein the term “substantially protective cellular response”refers to an immune response that is mediated by T-lymphocytes and/orother white blood cells against influenza virus, exhibited by avertebrate (e.g., a human), that prevents or ameliorates influenzainfection or reduces at least one symptom thereof. One important aspectof cellular immunity involves an antigen-specific response by cytolyticT-cells (“CTL”s). CTLs have specificity for peptide antigens that arepresented in association with proteins encoded by the majorhistocompatibility complex (MHC) and expressed on the surfaces of cells.CTLs help induce and promote the destruction of intracellular microbes,or the lysis of cells infected with such microbes. Another aspect ofcellular immunity involves an antigen-specific response by helperT-cells. Helper T-cells act to help stimulate the function, and focusthe activity of, nonspecific effector cells against cells displayingpeptide antigens in association with MHC molecules on their surface. A“cellular immune response” also refers to the production of cytokines,chemokines and other such molecules produced by activated T-cells and/orother white blood cells, including those derived from CD4+ and CD8+T-cells.

As used herein the term “substantial immunity in a population-widebasis” refers to immunity as a result of VLPs of the inventionadministered to individuals in a population. The immunity in saidindividual in said population results in the prevention, amelioration ofinfluenza infection, or reduction of at least one symptom related toinfluenza virus infection in said individual, and prevents the spread ofsaid influenza virus to others in the population. The term population isdefined as group of individuals (e.g. schoolchildren, elderly, healthyindividuals etc.) and may comprise a geographic area (e.g. specificcities, schools, neighborhoods, workplace, country, state, etc.).

As use herein, the term “antigenic formulation” or “antigeniccomposition” refers to a preparation which, when administered to avertebrate, especially a bird or a mammal, will induce an immuneresponse.

As use herein, the term “vertebrate” or “subject” or “patient” refers toany member of the subphylum cordata, including, without limitation,humans and other primates, including non-human primates such aschimpanzees and other apes and monkey species. Farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs; birds, including domestic, wild and game birds such as chickens,turkeys and other gallinaceous birds, ducks, geese, and the like arealso non-limiting examples. The terms “mammals” and “animals” areincluded in this definition. Both adult and newborn individuals areintended to be covered.

Influenza remains a pervasive public health concern despite theavailability of specific inactivated virus vaccines that are 60-80%effective under optimal conditions. When these vaccines are effective,illness is usually averted by preventing viral infection. Vaccinefailure can occur as a result of accumulated antigenic differences(antigenic shift and antigenic drift). For example, avian influenzavirus type A H9N2 co-circulated with human influenza virus type ASydney/97 (H3N2) in pigs and led to genetic reassortment and emergenceof new strains of human influenza virus with pandemic potential (Peiriset al., 2001). In the event of such antigenic shift, it is unlikely thatcurrent vaccines would provide adequate protection.

Another reason for the paucity of influenza vaccine programs is therelatively short persistence of immunity elicited by the currentvaccines. Further inadequacy of influenza control measures reflectsrestricted use of current vaccines because of vaccine reactogenicity andside effects in young children, elderly, and people with allergies tocomponents of eggs, which are used in manufacturing of commerciallylicensed inactivated virus influenza vaccines.

Additionally, inactivated influenza virus vaccines often lack or containaltered HA and NA conformational epitopes, which elicit neutralizingantibodies and play a major role in protection against disease. Thus,inactivated viral vaccines, as well as some recombinant monomericinfluenza subunit protein vaccines, deliver inadequate protection. Onthe other hand, macromolecular protein structures, such as capsomers,subviral particles, and/or VLPs, include multiple copies of nativeproteins exhibiting conformational epitopes, which are advantageous foroptimal vaccine immunogenicity.

The present invention describes the cloning of avian influenza A/HongKong/1073/99 (H9N2) virus HA, NA, and M1 genes into a single baculovirusexpression vector alone or in tandem and production of influenza vaccinecandidates or reagents comprised of recombinant influenza structuralproteins that self-assemble into functional and immunogenic homotypicmacromolecular protein structures, including subviral influenzaparticles and influenza VLP, in baculovirus-infected insect cells.

The present invention describes the cloning of human influenzaA/Sydney/5/97 and A/Fujian/411/2002 (H3N2) virus HA, NA, M1, M2, and NPgenes into baculovirus expression vectors and production influenzavaccine candidates or reagents comprised of influenza structuralproteins that self-assemble into functional and immunogenic homotypicmacromolecular protein structures, including subviral influenzaparticles and influenza VLP, in baculovirus-infected insect cells.

In addition, the instant invention describes the cloning of the HA geneof human influenza A/Sydney/5/97 and A/Fujian/411/2002 (H3N2) virus andthe HA, NA, and M1 genes of avian influenza A/Hong Kong/1073/99 (H9N2)into a single baculovirus expression vector in tandem and productioninfluenza vaccine candidates or reagents comprised of influenzastructural proteins that self-assemble into functional and immunogenicheterotypic macromolecular protein structures, including subviralinfluenza particles and influenza VLP, in baculovirus-infected insectcells.

VLPs of the Invention

Influenza VLPs of the invention are useful for preparing vaccinesagainst influenza viruses. One important feature of this system is theability to replace the surface glycoproteins with different subtypes ofHA and/or NA or other viral proteins, thus, allowing updating of newinfluenza antigenic variants every year or to prepare for an influenzapandemic. As antigenic variants of these glycoproteins are identified,the VLPs can be updated to include these new variants (e.g. for seasonalinfluenza vaccines). In addition, surface glycoproteins from potentiallypandemic viruses, such as H5N1, or other HA, NA combinations withpandemic potential could be incorporated into VLPs without concern ofreleasing genes that had not circulated in humans for several decades.This is because the VLPs are not infectious, do not replicate and cannotcause disease. Thus, this system allows for creating a new candidateinfluenza vaccine every year and/or an influenza pandemic vaccinewhenever it is necessary.

There are 16 different hemagglutinin (HA) and 9 different neuraminidase(NA) all of which have been found among wild birds. Wild birds are theprimary natural reservoir for all types of influenza A viruses and arethought to be the source of all types of influenza A viruses in allother vertebrates. These subtypes differ because of changes in thehemagglutinin (HA) and neuraminidase (NA) on their surface. Manydifferent combinations of HA and NA proteins are possible. Eachcombination represents a different type of influenza A virus. Inaddition, each type can be further classified into strains based ondifferent mutations found in each of its 8 genes.

All known types of influenza A viruses can be found in birds. Usuallyavian influenza viruses do not infect humans. However, some avianinfluenza viruses develop genetic variations associated with thecapability of crossing the species barrier. Such a virus is capable ofcausing a pandemic because humans have no natural immunity to the virusand can easily spread from person to person. In 1997, avian influenzavirus jumped from a bird to a human in Hong Kong during an outbreak ofbird flu in poultry. This virus was identified as influenza virus H5N1.The virus caused severe respiratory illness in 18 people, six of whomdied. Since that time, many more cases of known H5N1 infections haveoccurred among humans worldwide; approximately half of those people havedied.

Thus, the present invention encompasses the cloning of HA, NA and M1nucleotides from avian influenza viruses, influenza viruses withpandemic potential and/or seasonal influenza viruses into expressionvectors. The present invention also describes the production ofinfluenza vaccine candidates or reagents comprised of influenza proteinsthat self-assemble into functional VLPs. All combinations of viralproteins must be co-expressed with a M1 nucleotide.

VLPs of the invention consist or comprise influenza HA, NA and M1proteins. In one embodiment, said VLP comprises a HA from an avian,pandemic and/or seasonal influenza virus and a NA from an avian,pandemic and/or seasonal influenza virus, wherein said HA is selectedfrom the group consisting of H1, H2, H3, H4, H5, H6, H, 7H8, H9, H10,H11, H12, H13, H14, H15 and H16 and said NA is selected from the groupconsisting of N1, N2, N3, N4, N5, N6, N7, N8 and N9. In anotherembodiment, the invention comprises a VLP that consists essentially ofHA, NA and M1. Said HA and NA can be from the above list of HA and NA.These VLPs may comprise additional influenza proteins and/or proteincontaminates in negligible concentrations. In another embodiment, saidinfluenza VLP comprises influenza proteins, wherein said influenzaproteins consist of HA, NA and M1 proteins. These VLPs contain HA, NAand M1 and may contain additional cellular constituents such as cellularproteins, baculovirus proteins, lipids, carbohydrates etc., but do notcontain additional influenza proteins (other than fragments of M1, HAand/or NA). In another embodiment, the HA and/or the NA may exhibithemagglutinin activity and/or neuraminidase activity, respectively, whenexpressed on the surface of VLPs.

In another embodiment, said VLP comprises HA and NA of the H5N1 virusand a M1 protein (the M1 protein may or may not be from the same viralstrain). In another embodiment, said VLP consists essentially of HA, NAof the H5N1 virus and a M1 protein. These VLPs may comprise additionalinfluenza proteins and/or protein contaminates in negligibleconcentrations. In a further embodiment, said VLP consists of HA, NA ofthe H5N1 virus and a M1 protein. In another embodiment, said influenzaVLP comprises influenza proteins, wherein said influenza proteinsconsist of H5, N1 and M1 proteins. These VLPs contain H5, N9 and M1 andmay contain additional cellular constituents such as cellular proteins,baculovirus proteins, lipids, carbohydrates etc., but do not containadditional influenza proteins (other than fragments of M1, H5 and/orN1). In another embodiment, the H5 and/or the N1 may exhibithemagglutinin activity and/or neuraminidase activity, respectively, whenexpressed on the surface of VLPs.

In another embodiment, said VLP comprises the HA and NA of the H9N2virus, and a M1 protein. In another embodiment, said VLP consistsessentially of the HA and NA of the H9N2 virus, and a M1 protein. TheseVLPs may comprise additional influenza proteins and/or proteincontaminates in negligible concentrations. In another embodiment, saidVLP consists of the HA and NA of the H9N2 virus, and a M1 protein. Inanother embodiment, said influenza VLP comprises influenza proteins,wherein said influenza proteins consist of H9, N2 and M1 proteins. TheseVLPs contain H9, N2 and M1 and may contain additional cellularconstituents such as cellular proteins, baculovirus proteins, lipids,carbohydrates etc., but do not contain additional influenza proteins(other than fragments of M1, H9 and/or N2). In another embodiment, theH9 and/or the N2 may exhibit hemagglutinin activity and/or neuraminidaseactivity, respectively, when expressed on the surface of VLPs.

In another embodiment, said VLP comprises the HA and NA from aninfluenza B virus, and a M1 protein. Influenza B viruses are usuallyfound only in humans. Unlike influenza A viruses; these viruses are notclassified according to subtype. Influenza B viruses can cause morbidityand mortality among humans, but in general are associated with lesssevere epidemics than influenza A viruses. In another embodiment, saidVLP consists essentially of the HA and NA of the influenza B virus, anda M1 protein. These VLPs may comprise additional influenza proteinsand/or protein contaminates in negligible concentrations. In anotherembodiment, said influenza VLP comprises influenza proteins, whereinsaid influenza proteins consist of HA, NA and M1 proteins. These VLPscontain HA, NA and M1 and may contain additional cellular constituentssuch as cellular proteins, baculovirus proteins, lipids, carbohydratesetc., but do not contain additional influenza proteins (other thanfragments of M1, HA and/or NA). In another embodiment, said VLP consistsof the HA and NA of the influenza B virus, and a M1 protein. In anotherembodiment, the HA and/or the NA may exhibit hemagglutinin activityand/or neuraminidase activity, respectively, when expressed on thesurface of VLPs.

The invention also encompasses variants of the said influenza proteinsexpressed on or in the VLPs of the invention. The variants may containalterations in the amino acid sequences of the constituent proteins. Theterm “variant” with respect to a polypeptide refers to an amino acidsequence that is altered by one or more amino acids with respect to areference sequence. The variant can have “conservative” changes, whereina substituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine. Alternatively, a variantcan have “nonconservative” changes, e.g., replacement of a glycine witha tryptophan. Analogous minor variations can also include amino aciddeletion or insertion, or both. Guidance in determining which amino acidresidues can be substituted, inserted, or deleted without eliminatingbiological or immunological activity can be found using computerprograms well known in the art, for example, DNASTAR software.

Natural variants can occur due to antigenic drifts. Antigenic drifts aresmall changes in the viral proteins that happen continually over time.Thus, a person infected with a particular flu virus strain developsantibody against that virus, as newer virus strains appear, theantibodies against the older strains no longer recognize the newer virusand reinfection can occur. This is why there is a new vaccine forinfluenza each season. In addition, some changes in an influenza viruscan cause influenza virus to cross species. For example, some avianinfluenza viruses developed genetic variations associated with thecapability of crossing the species barrier. Such a virus is capable ofcausing a pandemic because people have no natural immunity to the virusand the virus can easily spread from person to person. Thesenaturallyoccurring variations of the influenza proteins are anembodiment of the invention.

General texts which describe molecular biological techniques, which areapplicable to the present invention, such as cloning, mutation, cellculture and the like, include Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymology volume 152 Academic Press,Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—ALaboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 2000 (“Sambrook”) and Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., (“Ausubel”). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, e.g., thecloning and mutation of HA and/or NA molecules, etc. Thus, the inventionalso encompasses using known methods of protein engineering andrecombinant DNA technology to improve or alter the characteristics ofthe influenza proteins expressed on or in the VLPs of the invention.Various types of mutagenesis can be used to produce and/or isolatevariant HA, NA and/or M1 molecules and/or to further modify/mutate thepolypeptides of the invention. They include but are not limited tosite-directed, random point mutagenesis, homologous recombination (DNAshuffling), mutagenesis using uracil containing templates,oligonucleotide-directed mutagenesis, phosphorothioate-modified DNAmutagenesis, mutagenesis using gapped duplex DNA or the like. Additionalsuitable methods include point mismatch repair, mutagenesis usingrepair-deficient host strains, restriction-selection andrestriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,e.g., involving chimeric constructs, is also included in the presentinvention. In one embodiment, mutagenesis can be guided by knowninformation of the naturally occurring molecule or altered or mutatednaturally occurring molecule, e.g., sequence, sequence comparisons,physical properties, crystal structure or the like.

The invention further comprises influenza protein variants which showsubstantial biological activity, e.g., able to elicit an effectiveantibody response when expressed on or in a VLP. Such variants includedeletions, insertions, inversions, repeats, and substitutions selectedaccording to general rules known in the art so as have little effect onactivity.

Methods of cloning said influenza proteins are known in the art. Forexample, the influenza gene encoding a specific influenza protein can beisolated by RT-PCR from polyadenylated mRNA extracted from cells whichhad been infected with an influenza virus. The resulting product genecan be cloned as a DNA insert into a vector. The term “vector” refers tothe means by which a nucleic acid can be propagated and/or transferredbetween organisms, cells, or cellular components. Vectors includeplasmids, viruses, bacteriophages, pro-viruses, phagemids, transposons,artificial chromosomes, and the like, that replicate autonomously or canintegrate into a chromosome of a host cell. A vector can also be a nakedRNA polynucleotide, a naked DNA polynucleotide, a polynucleotidecomposed of both DNA and RNA within the same strand, apoly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, aliposome-conjugated DNA, or the like, that is not autonomouslyreplicating. In many, but not all, common embodiments, the vectors ofthe present invention are plasmids or bacmids.

Thus, the invention comprises nucleotides which encode the HA, NA and/orM1 influenza proteins cloned into an expression vector which can beexpressed in a cell which induces the formation of VLPs. An “expressionvector” is a vector, such as a plasmid that is capable of promotingexpression, as well as replication of a nucleic acid incorporatedtherein. Typically, the nucleic acid to be expressed is “operablylinked” to a promoter and/or enhancer, and is subject to transcriptionregulatory control by the promoter and/or enhancer. In one embodiment,said nucleotides that encode for HA from an avian, pandemic and/orseasonal influenza virus is selected from the group consisting of H1,H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16. Inanother embodiment, said nucleotides that encode for NA from an avian,pandemic and/or seasonal influenza virus, is selected from the groupconsisting of N1, N2, N3, N4, N5, N6, N7, N8 and N9. In anotherembodiment, said vector comprises of nucleotides that encode the HA, NAand/or M1 influenza protein. In another embodiment, said vector consistsof nucleotides that encodes the HA, NA and M1 influenza protein. Apreferred expression vector is a baculovirus vector. After thenucleotides encoding said influenza proteins have been cloned saidnucleotides can be further manipulated. For example, a person with skillin the art can mutate specific bases in the coding region to producevariants. The variants may contain alterations in the coding regions,non-coding regions, or both. Such variants may increase theimmunogenticity of an influenza protein or remove a splice site from aprotein or RNA. For example, in one embodiment, the donor and acceptorsplicing sites on the influenza M protein (full length) are mutated toprevent splicing of the M mRNA into M1 and M2 transcripts. In anotherembodiment the HA is engineered to remove or mutate the cleavage site.For example, wild type H5 HA has a cleavage site that contains multiplebasic amino acids (RRRKR). This wild type sequence makes the HA moresusceptible to multiple ubiquitous proteases that may be present in hostor system expression these HAs. In one embodiment, removing these aminoacids can reduce the susceptibility of the HA to various proteases. Inanother embodiment, the cleavage site can be mutated to remove thecleavage site (e.g. mutate to RESR).

The invention also utilizes nucleic acid and polypeptides which encodeNA, HA and M1. In one embodiment, an influenza NA nucleic acid orprotein is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQID NOs 1, 11, 31, 32, 39, 38, 46, 47, 54 or 55. In another embodiment,an influenza HA nucleic acid or protein is at least 85%, 90%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NOs 2, 10, 56, 57, 58, 27, 28, 29,30, 37, 36, 33, 34, 35, 42, 43, 44, 45, 50, 51, 52, or 53. In anotherembodiment, an influenza M1 nucleic acid or protein is at least 85%,90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs 12, 40, 41, 48 or49.

In some embodiments, mutations containing alterations which producesilent substitutions, additions, or deletions, but do not alter theproperties or activities of the encoded protein or how the proteins aremade. Nucleotide variants can be produced for a variety of reasons,e.g., to optimize codon expression for a particular host (change codonsin the human mRNA to those preferred by insect cells such as Sf9 cells).See U.S. patent publication 2005/0118191, herein incorporated byreference in its entirety for all purposes. Examples of optimized codonsequences of the invention are disclosed below (e.g. SEQ ID 42, 44, 46,48, 50, 52, and 54).

In addition, the nucleotides can be sequenced to ensure that the correctcoding regions were cloned and do not contain any unwanted mutations.The nucleotides can be subcloned into an expression vector (e.g.baculovirus) for expression in any cell. The above is only one exampleof how the influenza viral proteins can be cloned. A person with skillin the art understands that additional methods are available and arepossible.

The invention also provides for constructs and/or vectors that compriseavian, pandemic and/or seasonal nucleotides which encode for influenzavirus structural genes, including NA, M1 and/or HA. The vector may be,for example, a phage, plasmid, viral, or retroviral vector. Theconstructs and/or vectors that encodes avian, pandemic and/or seasonalinfluenza virus structural genes, including NA, M1 and/or HA should beoperatively linked to an appropriate promoter, such as the AcMNPVpolyhedrin promoter (or other baculovirus), phage lambda PL promoter,the E. coli lac, phoA and tac promoters, the SV40 early and latepromoters, and promoters of retroviral LTRs are non-limiting examples.Other suitable promoters will be known to the skilled artisan dependingon the host cell and/or the rate of expression desired. The expressionconstructs will further contain sites for transcription initiation,termination, and, in the transcribed region, a ribosome binding site fortranslation. The coding portion of the transcripts expressed by theconstructs will preferably include a translation initiating codon at thebeginning and a termination codon appropriately positioned at the end ofthe polypeptide to be translated.

The expression vectors will preferably include at least one selectablemarker. Such markers include dihydrofolate reductase, G418 or neomycinresistance for eukaryotic cell culture and tetracycline, kanamycin orampicillin resistance genes for culturing in E. coli and other bacteria.Among vectors preferred are virus vectors, such as baculovirus, poxvirus(e.g., vaccinia virus, avipox virus, canarypox virus, fowlpox virus,raccoonpox virus, swinepox virus, etc.), adenovirus (e.g., canineadenovirus), herpesvirus, and retrovirus. Other vectors that can be usedwith the invention comprise vectors for use in bacteria, which comprisepQE70, pQE60 and pQE-9, pBluescript vectors, Phagescript vectors, pNH8A,pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5.Among preferred eukaryotic vectors are pFastBac1 pWINEO, pSV2CAT, pOG44,pXT1 and pSG, pSVK3, pBPV, pMSG, and pSVL. Other suitable vectors willbe readily apparent to the skilled artisan. In one embodiment, saidvector that comprises nucleotides encoding for avian, pandemic and/orseasonal influenza virus structural genes, including HA, M1 and/or NA,is pFastBac. In another embodiment, said vector that comprises an insertthat consists of nucleotides encoding for avian, pandemic and/orseasonal influenza virus structural genes, comprises HA, M1 and NA, ispFastBac.

Next, the recombinant vector can be transfected, infected, ortransformed into a suitable host cell. Thus, the invention provides forhost cells which comprise a vector (or vectors) that contain nucleicacids which code for HA, M1 and/or NA and permit the expression of HA,M1 and/or NA in said host cell under conditions which allow theformation of VLPs.

In one embodiment, the recombinant constructs mentioned above could beused to transfect, infect, or transform and can express HA, NA and M1influenza proteins in eukaryotic cells and/or prokaryotic cells. Amongeukaryotic host cells are yeast, insect, avian, plant, C. elegans (ornematode) and mammalian host cells. Non limiting examples of insectcells are, Spodoptera frugiperda (Sf) cells, e.g. Sf9, Sf21,Trichoplusia ni cells, e.g. High Five cells; and Drosophila S2 cells.Examples of fungi (including yeast) host cells are S. cerevisiae,Kluyveromyces lactis (K. lactis), species of Candida including C.albicans and C. glabrata, Aspergillus nidulans, Schizosaccharomycespombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica. Examples ofmammalian cells are COS cells, baby hamster kidney cells, mouse L cells,LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney(HEK) cells, and African green monkey cells, CV1 cells, HeLa cells, MDCKcells, Vero and Hep-2 cells. Xenopus laevis oocytes, or other cells ofamphibian origin, may also be used. Prokaryotic host cells includebacterial cells, for example, E. coli, B. subtilis, and mycobacteria.

Vectors, e.g., vectors comprising HA, NA and/or M1 polynucleotides, canbe transfected into host cells according to methods well known in theart. For example, introducing nucleic acids into eukaryotic cells can beby calcium phosphate co-precipitation, electroporation, microinjection,lipofection, and transfection employing polyamine transfection reagents.In one embodiment, the said vector is a recombinant baculovirus. Inanother embodiment, said recombinant baculovirus is transfected into aeukaryotic cell. In a preferred embodiment, said cell is an insect cell.In another embodiment, said insect cell is a Sf9 cell.

In another embodiment, said vector and/or host cell comprise nucleotideswhich encode an avian, pandemic and/or seasonal influenza virus HAprotein selected from the group consisting of H1, H2, H3, H4, H5, H6,H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16. In another embodiment,said vector and/or host cells comprise nucleotides which encode an NAprotein which is selected from the group consisting of N1, N2, N3, N4,N5, N6, N7, N8 and N9. In another embodiment, said vector and/or hostcell comprises influenza HA, M1 and/or NA. In another embodiment, saidvector and/or host cell consists essentially of HA, M1 and/or NA. In afurther embodiment, said vector and/or host cell consists of influenzaprotein comprising HA, M1 and NA. These vector and/or host cell containHA, NA and M1 and may contain additional cellular constituents such ascellular proteins, baculovirus proteins, lipids, carbohydrates etc., butdo not contain additional influenza proteins (other than fragments ofM1, HA and/or NA). In another embodiment, said nucleotides encode for anHA and/or the NA that exhibits hemagglutinin activity and/orneuraminidase activity, respectively, when expressed on the surface ofVLPs.

This invention also provides for constructs and methods that willincrease the efficiency of VLPs production. For example, removingcleavage sites from proteins in order to increase protein expression(see above). Other method comprises the addition of leader sequences tothe HA, NA and/or M1 protein for more efficient transporting. Forexample, a heterologous signal sequence can be fused to the HA, NAand/or M1 influenza protein. In one embodiment, the signal sequence canbe derived from the gene of an insect cell and fused to the influenza HAprotein (for expression in insect cells). In another embodiment, thesignal peptide is the chitinase signal sequence, which works efficientlyin baculovirus expression systems. In other embodiment, interchangingleader sequences between influenza proteins can provide better proteintransport. For example, it has been shown that H5 hemagglutinin is lessefficient at being transported to the surface of particles. H9hemagglutinins, however, targets the surface and is integrated into thesurface more efficiently. Thus, in one embodiment, the H9 leadersequence is fused to the H5 protein.

Another method to increase efficiency of VLP production is to codonoptimize the nucleotides that encode HA, NA and/or M1 proteins for aspecific cell type. For example, codon optimizing nucleic acids forexpression in Sf9 cell (see U.S. patent publication 2005/0118191, hereinincorporated by reference in its entirety for all purposes). Examples ofoptimized codon sequences for Sf9 cells are disclosed below (e.g. SEQ ID42, 44, 46, 48, 50, 52, and 54). In one embodiment, the nucleic acidsequence of codon optimized influenza protein is at least 85%, 90%, 95%,96, 97, 98, or 99% to any one of SEQ ID Nos. 42, 44, 46, 48, 50, 52, and54.

The invention also provides for methods of producing VLPs, said methodscomprising expressing an avian, pandemic and/or seasonal influenzaproteins under conditions that allow VLP formation. Depending on theexpression system and host cell selected, the VLPs are produced bygrowing host cells transformed by an expression vector under conditionswhereby the recombinant proteins are expressed and VLPs are formed. Theselection of the appropriate growth conditions is within the skill or aperson with skill of one of ordinary skill in the art.

Methods to grow cells engineered to produce VLPs of the inventioninclude, but are not limited to, batch, batch-fed, continuous andperfusion cell culture techniques. Cell culture means the growth andpropagation of cells in a bioreactor (a fermentation chamber) wherecells propagate and express protein (e.g. recombinant proteins) forpurification and isolation. Typically, cell culture is performed understerile, controlled temperature and atmospheric conditions in abioreactor. A bioreactor is a chamber used to culture cells in whichenvironmental conditions such as temperature, atmosphere, agitationand/or pH can be monitored. In one embodiment, said bioreactor is astainless steel chamber. In another embodiment, said bioreactor is apre-sterilized plastic bag (e.g. Cellbag®, Wave Biotech, Bridgewater,N.J.). In other embodiment, said pre-sterilized plastic bags are about50 L to 1000 L bags.

The VLPs are then isolated using methods that preserve the integritythereof, such as by gradient centrifugation, e.g., cesium chloride,sucrose and iodixanol, as well as standard purification techniquesincluding, e.g., ion exchange and gel filtration chromatography.

The following is an example of how VLPs of the invention can be made,isolated and purified. Usually VLPs are produced from recombinant celllines engineered to create a VLP when said cells are grown in cellculture (see above). Production of VLPs may be accomplished by thescheme illustrated in FIG. 26. A person of skill in the art wouldunderstand that there are additional methods that can be utilized tomake and purify VLPs of the invention, thus the invention is not limitedto the method described.

Production of VLPs of the invention can start by seeding Sf9 cells(non-infected) into shaker flasks, allowing the cells to expand andscaling up as the cells grow and multiply (for example from a 125-mlflask to a 50 L Wave bag). The medium used to grow the cell isformulated for the appropriate cell line (preferably serum free media,e.g. insect medium ExCell-420, JRH). Next, said cells are infected withrecombinant baculovirus at the most efficient multiplicity of infection(e.g. from about 1 to about 3 plaque forming units per cell). Onceinfection has occurred, the influenza HA, NA and M1 proteins areexpressed from the virus genome, self assemble into VLPs and aresecreted from the cells approximately 24 to 72 hours post infection.Usually, infection is most efficient when the cells are in mid-log phaseof growth (4−8×10⁶ cells/ml) and are at least about 90% viable.

VLPs of the invention can be harvested approximately 48 to 96 hours postinfection, when the levels of VLPs in the cell culture medium are nearthe maximum but before extensive cell lysis. The Sf9 cell density andviability at the time of harvest can be about 0.5×10⁶ cells/ml to about1.5×10⁶ cells/ml with at least 20% viability, as shown by dye exclusionassay. Next, the medium is removed and clarified. NaCl can be added tothe medium to a concentration of about 0.4 to about 1.0 M, preferably toabout 0.5 M, to avoid VLP aggregation. The removal of cell and cellulardebris from the cell culture medium containing VLPs of the invention canbe accomplished by tangential flow filtration (TFF) with a single use,pre-sterilized hollow fiber 0.5 or 1.00 μm filter cartridge or a similardevice.

Next, VLPs in the clarified culture medium can be concentrated byultrafiltration using a disposable, pre-sterilized 500,000 molecularweight cut off hollow fiber cartridge. The concentrated VLPs can bediafiltrated against 10 volumes pH 7.0 to 8.0 phosphate-buffered saline(PBS) containing 0.5 M NaCl to remove residual medium components.

The concentrated, diafiltered VLPs can be furthered purified on a 20% to60% discontinuous sucrose gradient in pH 7.2 PBS buffer with 0.5 M NaClby centrifugation at 6,500×g for 18 hours at about 4° C. to about 10° C.Usually VLPs will form a distinctive visible band between about 30% toabout 40% sucrose or at the interface (in a 20% and 60% step gradient)that can be collected from the gradient and stored. This product can bediluted to comprise 200 mM of NaCl in preparation for the next step inthe purification process. This product contains VLPs and may containintact baculovirus particles.

Further purification of VLPs can be achieved by anion exchangechromatography, or 44% isopycnic sucrose cushion centrifugation. Inanion exchange chromatography, the sample from the sucrose gradient (seeabove) is loaded into column containing a medium with an anion (e.g.Matrix Fractogel EMD TMAE) and eluded via a salt gradient (from about0.2 M to about 1.0 M of NaCl) that can separate the VLP from othercontaminates (e.g. baculovirus and DNA/RNA). In the sucrose cushionmethod, the sample comprising the VLPs is added to a 44% sucrose cushionand centrifuged for about 18 hours at 30,000 g. VLPs form a band at thetop of 44% sucrose, while baculovirus precipitates at the bottom andother contaminating proteins stay in the 0% sucrose layer at the top.The VLP peak or band is collected.

The intact baculovirus can be inactivated, if desired. Inactivation canbe accomplished by chemical methods, for example, formalin or β-propyllactone (BPL). Removal and/or inactivation of intact baculovirus canalso be largely accomplished by using selective precipitation andchromatographic methods known in the art, as exemplified above. Methodsof inactivation comprise incubating the sample containing the VLPs in0.2% of BPL for 3 hours at about 25° C. to about 27° C. The baculoviruscan also be inactivated by incubating the sample containing the VLPs at0.05% BPL at 4° C. for 3 days, then at 37° C. for one hour.

After the inactivation/removal step, the product comprising VLPs can berun through another diafiltration step to remove any reagent from theinactivation step and/or any residual sucrose, and to place the VLPsinto the desired buffer (e.g. PBS). The solution comprising VLPs can besterilized by methods known in the art (e.g. sterile filtration) andstored in the refrigerator or freezer.

The above techniques can be practiced across a variety of scales. Forexample, T-flasks, shake-flasks, spinner bottles, up to industrial sizedbioreactors. The bioreactors can comprise either a stainless steel tankor a pre-sterilized plastic bag (for example, the system sold by WaveBiotech, Bridgewater, N.J.). A person with skill in the art will knowwhat is most desirable for their purposes.

Expansion and production of baculovirus expression vectors and infectionof cells with recombinant baculovirus to produce recombinant influenzaVLPs can be accomplished in insect cells, for example Sf9 insect cellsas previously described. In a preferred embodiment, the cells are SF9infected with recombinant baculovirus engineered to produce influenzaVLPs.

Pharmaceutical or Vaccine Formulations and Administration

The pharmaceutical compositions useful herein contain a pharmaceuticallyacceptable carrier, including any suitable diluent or excipient, whichincludes any pharmaceutical agent that does not itself induce theproduction of an immune response harmful to the vertebrate receiving thecomposition, and which may be administered without undue toxicity and aVLP of the invention. As used herein, the term “pharmaceuticallyacceptable” means being approved by a regulatory agency of the Federalor a state government or listed in the U.S. Pharmacopia, EuropeanPharmacopia or other generally recognized pharmacopia for use invertebrates, and more particularly in humans. These compositions can beuseful as a vaccine and/or antigenic compositions for inducing aprotective immune response in a vertebrate.

Said pharmaceutical formulations of the invention comprise VLPscomprising an influenza M1, HA and/or NA protein and a pharmaceuticallyacceptable carrier or excipient. Pharmaceutically acceptable carriersinclude but are not limited to saline, buffered saline, dextrose, water,glycerol, sterile isotonic aqueous buffer, and combinations thereof. Athorough discussion of pharmaceutically acceptable carriers, diluents,and other excipients is presented in Remington's Pharmaceutical Sciences(Mack Pub. Co. N.J. current edition). The formulation should suit themode of administration. In a preferred embodiment, the formulation issuitable for administration to humans, preferably is sterile,non-particulate and/or non-pyrogenic.

The composition, if desired; can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be asolid form, such as a lyophilized powder suitable for reconstitution, aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc.

The invention also provides for a pharmaceutical pack or kit comprisingone or more containers filled with one or more of the ingredients of thevaccine formulations of the invention. In a preferred embodiment, thekit comprises two containers, one containing VLPs and the othercontaining an adjuvant. Associated with such container(s) can be anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

The invention also provides that the VLP formulation be packaged in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of composition. In one embodiment, the VLP composition issupplied as a liquid, in another embodiment, as a dry sterilizedlyophilized powder or water free concentrate in a hermetically sealedcontainer and can be reconstituted, e.g., with water or saline to theappropriate concentration for administration to a subject. Preferably,the VLP composition is supplied as a dry sterile lyophilized powder in ahermetically sealed container at a unit dosage of preferably, about 1μg, about 5 μg, about 10 μg, about 20 μg, about 25 μg, about 30 μg,about 50 μg, about 100 μg, about 125 μg, about 150 μg, or about 200 μg.Alternatively, the unit dosage of the VLP composition is less than about1 μg, (for example about 0.08 μg, about 0.04 μg; about 0.2 μg, about 0.4μg, about 0.8 μg, about 0.5 μg or less, about 0.25 μg or less, or about0.1 μg or less), or more than about 125 μg, (for example about 150 μg ormore, about 250 μg or more, or about 500 μg or more). These doses may bemeasured as total VLPs or as μg of HA. The VLP composition should beadministered within about 12 hours, preferably within about 6 hours,within about 5 hours, within about 3 hours, or within about 1 hour afterbeing reconstituted from the lyophylized powder.

In an alternative embodiment, a VLP composition is supplied in liquidform in a hermetically sealed container indicating the quantity andconcentration of the VLP composition. Preferably, the liquid form of theVLP composition is supplied in a hermetically sealed container at leastabout 50 μg/ml, more preferably at least about 100 μg/ml, at least about200 μg/ml, at least 500 μg/ml, or at least 1 mg/ml.

Generally, influenza VLPs of the invention are administered in aneffective amount or quantity (as defined above) sufficient to stimulatean immune response against one or more strains of influenza virus.Preferably, administration of the VLP of the invention elicitssubstantial immunity against at least one influenza virus. Typically,the dose can be adjusted within this range based on, e.g., age, physicalcondition, body weight, sex, diet, time of administration, and otherclinical factors. The prophylactic vaccine formulation is systemicallyadministered, e.g., by subcutaneous or intramuscular injection using aneedle and syringe, or a needle-less injection device. Alternatively,the vaccine formulation is administered intranasally, either by drops,large particle aerosol (greater than about 10 microns), or spray intothe upper respiratory tract. While any of the above routes of deliveryresults in an immune response, intranasal administration confers theadded benefit of eliciting mucosal immunity at the site of entry of theinfluenza virus.

Thus, the invention also comprises a method of formulating a vaccine orantigenic composition that induces substantial immunity to influenzavirus infection or at least one symptom thereof to a subject, comprisingadding to said formulation an effective dose of an influenza VLP.

While stimulation of substantial immunity with a single dose ispreferred, additional dosages can be administered, by the same ordifferent route, to achieve the desired effect. In neonates and infants,for example, multiple administrations may be required to elicitsufficient levels of immunity. Administration can continue at intervalsthroughout childhood, as necessary to maintain sufficient levels ofprotection against influenza infection. Similarly, adults who areparticularly susceptible to repeated or serious influenza infection,such as, for example, health care workers, day care workers, familymembers of young children, the elderly, and individuals with compromisedcardiopulmonary function may require multiple immunizations to establishand/or maintain protective immune responses. Levels of induced immunitycan be monitored, for example, by measuring amounts of neutralizingsecretory and serum antibodies, and dosages adjusted or vaccinationsrepeated as necessary to elicit and maintain desired levels ofprotection.

Thus, in one embodiment, a method to induce substantial immunity toinfluenza virus infection or at least one symptom thereof in a subject,comprises administering at least one effective dose of an influenza VLP,wherein said VLP comprises influenza HA, NA and M1 proteins. In anotherembodiment, a method of inducing substantial immunity to influenza virusinfection or at least one symptom thereof in a subject, comprisesadministering at least one effective dose of an influenza VLP, whereinsaid VLP consists essentially of influenza HA, NA and M1. Said VLPs maycomprise additional influenza proteins and/or protein contaminates innegligible concentrations. In another embodiment, a method of inducingsubstantial immunity to influenza virus infection or at least onesymptom thereof in a subject, comprises administering at least oneeffective dose of an influenza VLP, wherein said VLP consists ofinfluenza HA, NA and M1. In another embodiment, said influenza HA, NAand M1 is derived from seasonal influenza and/or avian influenza virus.In another embodiment, a method of inducing substantial immunity toinfluenza virus infection or at least one symptom thereof in a subject,comprises administering at least one effective dose of an influenza VLPcomprises influenza proteins, wherein said influenza proteins consist ofHA, NA and M1 proteins. These VLPs contain HA, NA and M1 and may containadditional cellular constituents such as cellular proteins, baculovirusproteins, lipids, carbohydrates etc., but do not contain additionalinfluenza proteins (other than fragments of M1, HA and/or NA). Inanother embodiment, said HA and/or NA exhibits hemagglutinin activityand/or neuraminidase activity, respectfully. In another embodiment, saidsubject is a mammal. In another embodiment, said mammal is a human. Inanother embodiment, the method comprises inducing substantial immunityto influenza virus infection or at least one symptom thereof byadministering said formulation in one dose. In another embodiment, themethod comprises inducing substantial immunity to influenza virusinfection or at least one symptom thereof by administering saidformulation in multiple doses.

Methods of administering a composition comprising VLPs (vaccine and/orantigenic formulations) include, but are not limited to, parenteraladministration (e.g., intradermal, intramuscular, intravenous andsubcutaneous), epidural, and mucosal (e.g., intranasal and oral orpulmonary routes or by suppositories). In a specific embodiment,compositions of the present invention are administered intramuscularly,intravenously, subcutaneously, transdermally or intradermally. Thecompositions may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucous, colon, conjunctiva,nasopharynx, oropharynx, vagina, urethra, urinary bladder and intestinalmucosa, etc.) and may be administered together with other biologicallyactive agents. In some embodiments, intranasal or other mucosal routesof administration of a composition comprising VLPs of the invention mayinduce an antibody or other immune response that is substantially higherthan other routes of administration. In another embodiment, intranasalor other mucosal routes of administration of a composition comprisingVLPs of the invention may induce an antibody or other immune responsethat will induce cross protection against other strains of influenzaviruses. Administration can be systemic or local.

In yet another embodiment, the vaccine and/or antigenic formulation isadministered in such a manner as to target mucosal tissues in order toelicit an immune response at the site of immunization. For example,mucosal tissues such as gut associated lymphoid tissue (GALT) can betargeted for immunization by using oral administration of compositionswhich contain adjuvants with particular mucosal targeting properties.Additional mucosal tissues can also be targeted, such as nasopharyngeallymphoid tissue (NALT) and bronchial-associated lymphoid tissue (BALT).

Vaccines and/or antigentic formulations of the invention may also beadministered on a dosage schedule, for example, an initialadministration of the vaccine composition with subsequent boosteradministrations. In particular embodiments, a second dose of thecomposition is administered anywhere from two weeks to one year,preferably from about 1, about 2, about 3, about 4, about 5 to about 6months, after the initial administration. Additionally, a third dose maybe administered after the second dose and from about three months toabout two years, or even longer, preferably about 4, about 5, or about 6months, or about 7 months to about one year after the initialadministration. The third dose may be optionally administered when no orlow levels of specific immunoglobulins are detected in the serum and/orurine or mucosal secretions of the subject after the second dose. In apreferred embodiment, a second dose is administered about one monthafter the first administration and a third dose is administered aboutsix months after the first administration. In another embodiment, thesecond dose is administered about six months after the firstadministration.

In another embodiment, said VLP of the invention can be administered aspart of a combination therapy. For example, VLPs of the invention can beformulated with other immunogenic compositions and/or antivirals (e.g.Amantadine, Rimantadine, Zanamivir and Osteltamivir).

The dosage of the pharmaceutical formulation can be determined readilyby the skilled artisan, for example, by first identifying doseseffective to elicit a prophylactic or therapeutic immune response, e.g.,by measuring the serum titer of virus specific immunoglobulins or bymeasuring the inhibitory ratio of antibodies in serum samples, or urinesamples, or mucosal secretions. Said dosages can be determined fromanimal studies. A non-limiting list of animals used to study theinfluenza virus include the guinea pig, Syrian hamster, chinchilla,hedgehog, chicken, rat, mouse and ferret. Most animals are not naturalhosts to influenza viruses but can still serve in studies of variousaspects of the disease. For example, any of the above animals can bedosed with a vaccine candidate, e.g. VLPs of the invention, to partiallycharacterize the immune response induced, and/or to determine if anyneutralizing antibodies have been produced. For example, many studieshave been conducted in the mouse model because mice are small size andtheir low cost allows researchers to conduct studies on a larger scale.Nevertheless, the mouse's small size also increases the difficulty ofreadily observing any clinical signs of the disease and the mouse is nota predictive model for disease in humans.

There has been extensive use of ferrets for studying various aspects ofhuman influenza viral infection and its course of action. Thedevelopment of many of the contemporary concepts of immunity to theinfluenza virus would have been impossible without the use of the ferret(Maher et al. 2004). Ferrets have proven to be a good model for studyinginfluenza for several reasons: influenza infection in the ferret closelyresembles that in humans with respect to clinical signs, pathogenesis,and immunity; types A and B of human influenza virus naturally infectthe ferret, thus providing an opportunity to study a completelycontrolled population in which to observe the interplay of transmissionof infection, illness, and sequence variation of amino acids in theglycoproteins of the influenza virus; and ferrets have other physicalcharacteristics that make it an ideal model for deciphering themanifestations of the disease. For example, ferrets and humans show verysimilar clinical signs of influenza infection that seem to depend on theage of the host, the strain of the virus, environmental conditions, thedegree of secondary bacterial infection, and many other variables. Thus,one skilled in the art can more easily correlate the efficacy of aninfluenza vaccine and dosage regiments from a ferret model to humans ascompared to a mouse or any other model described above.

In addition, human clinical studies can be performed to determine thepreferred effective dose for humans by a skilled artisan. Such clinicalstudies are routine and well known in the art. The precise dose to beemployed will also depend on the route of administration. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal test systems.

As also well known in the art, the immunogenicity of a particularcomposition can be enhanced by the use of non-specific stimulators ofthe immune response, known as adjuvants. Adjuvants have been usedexperimentally to promote a generalized increase in immunity againstunknown antigens (e.g., U.S. Pat. No. 4,877,611). Immunization protocolshave used adjuvants to stimulate responses for many years, and as such,adjuvants are well known to one of ordinary skill in the art. Someadjuvants affect the way in which antigens are presented. For example,the immune response is increased when protein antigens are precipitatedby alum. Emulsification of antigens also prolongs the duration ofantigen presentation. The inclusion of any adjuvant described in Vogelet al., “A Compendium of Vaccine Adjuvants and Excipients (2^(nd)Edition),” herein incorporated by reference in its entirety for allpurposes, is envisioned within the scope of this invention.

Exemplary, adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant. Other adjuvants comprise GMCSP, BCG, aluminum hydroxide, MDPcompounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, andmonophosphoryl lipid A (MPL). RIBI, which contains three componentsextracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wallskeleton (CWS) in a 2% squalene/Tween 80 emulsion also is contemplated.MF-59, Novasomes®, MHC antigens may also be used.

In one embodiment of the invention the adjuvant is a paucilamellar lipidvesicle having about two to ten bilayers arranged in the form ofsubstantially spherical shells separated by aqueous layers surrounding alarge amorphous central cavity free of lipid bilayers. Paucilamellarlipid vesicles may act to stimulate the immune response several ways, asnon-specific stimulators, as carriers for the antigen, as carriers ofadditional adjuvants, and combinations thereof. Paucilamellar lipidvesicles act as non-specific immune stimulators when, for example, avaccine is prepared by intermixing the antigen with the preformedvesicles such that the antigen remains extracellular to the vesicles. Byencapsulating an antigen within the central cavity of the vesicle, thevesicle acts both as an immune stimulator and a carrier for the antigen.In another embodiment, the vesicles are primarily made ofnonphospholipid vesicles. In other embodiment, the vesicles areNovasomes. Novasomes® are paucilamellar nonphospholipid vesicles rangingfrom about 100 nm to about 500 nm. They comprise Brij 72, cholesterol,oleic acid and squalene. Novasomes have been shown to be an effectiveadjuvant for influenza antigens (see, U.S. Pat. Nos. 5,629,021,6,387,373, and 4,911,928, herein incorporated by reference in theirentireties for all purposes).

In one aspect, an adjuvant effect is achieved by use of an agent, suchas alum, used in about 0.05 to about 0.1% solution in phosphate bufferedsaline. Alternatively, the VLPs can be made as an admixture withsynthetic polymers of sugars (Carbopol®) used as an about 0.25%solution. Some adjuvants, for example, certain organic moleculesobtained from bacteria; act on the host rather than on the antigen. Anexample is muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine[MDP]), a bacterial peptidoglycan. In other embodiments, hemocyanins andhemoerythrins may also be used with VLPs of the invention. The use ofhemocyanin from keyhole limpet (KLH) is preferred in certainembodiments, although other molluscan and arthropod hemocyanins andhemoerythrins may be employed.

Various polysaccharide adjuvants may also be used. For example, the useof various pneumococcal polysaccharide adjuvants on the antibodyresponses of mice has been described (Yin et al., 1989). The doses thatproduce optimal responses, or that otherwise do not produce suppression,should be employed as indicated (Yin et al., 1989). Polyamine varietiesof polysaccharides are particularly preferred, such as chitin andchitosan, including deacetylated chitin. In another embodiment, alipophilic disaccharide-tripeptide derivative of muramyl dipeptide whichis described for use in artificial liposomes formed from phosphatidylcholine and phosphatidyl glycerol.

Amphipathic and surface active agents, e.g., saponin and derivativessuch as QS21 (Cambridge Biotech), form yet another group of adjuvantsfor use with the VLPs of the invention. Nonionic block copolymersurfactants (Rabinovich et al., 1994) may also be employed.Oligonucleotides are another useful group of adjuvants (Yamamoto et al.,1988). Quil A and lentinen are other adjuvants that may be used incertain embodiments of the present invention.

Another group of adjuvants are the detoxified endotoxins, such as therefined detoxified endotoxin of U.S. Pat. No. 4,866,034. These refineddetoxified endotoxins are effective in producing adjuvant responses invertebrates. Of course, the detoxified endotoxins may be combined withother adjuvants to prepare multi-adjuvant formulation. For example,combination of detoxified endotoxins with trehalose dimycolate isparticularly contemplated, as described in U.S. Pat. No. 4,435,386.Combinations of detoxified endotoxins with trehalose dimycolate andendotoxic glycolipids is also contemplated (U.S. Pat. No. 4,505,899), asis combination of detoxified endotoxins with cell wall skeleton (CWS) orCWS and trehalose dimycolate, as described in U.S. Pat. Nos. 4,436,727,4,436,728 and 4,505,900. Combinations of just CWS and trehalosedimycolate, without detoxified endotoxins, is also envisioned to beuseful, as described in U.S. Pat. No. 4,520,019.

Those of skill in the art will know the different kinds of adjuvantsthat can be conjugated to vaccines in accordance with this invention andthese include alkyl lysophosphilipids (ALP); BCG; and biotin (includingbiotinylated derivatives) among others. Certain adjuvants particularlycontemplated for use are the teichoic acids from Gram-cells. Theseinclude the lipoteichoic acids (LTA), ribitol teichoic acids (RTA) andglycerol teichoic acid (GTA). Active forms of their syntheticcounterparts may also be employed in connection with the invention(Takada et al., 1995).

Various adjuvants, even those that are not commonly used in humans, maystill be employed in other vertebrates, where, for example, one desiresto raise antibodies or to subsequently obtain activated T cells. Thetoxicity or other adverse effects that may result from either theadjuvant or the cells, e.g., as may occur using non-irradiated tumorcells, is irrelevant in such circumstances.

Another method of inducing an immune response can be accomplished byformulating the VLPs of the invention with “immune stimulators.” Theseare the body's own chemical messengers (cytokines) to increase theimmune system's response. Immune stimulators include, but not limitedto, various cytokines, lymphokines and chemokines withimmunostimulatory, immunopotentiating, and pro-inflammatory activities,such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13);growth factors (e.g., granulocyte-macrophage (GM)-colony stimulatingfactor (CSF)); and other immunostimulatory molecules, such as macrophageinflammatory factor, Flt3 ligand, B7.1; B7.2, etc. The immunostimulatorymolecules can be administered in the same formulation as the influenzaVLPs, or can be administered separately. Either the protein or anexpression vector encoding the protein can be administered to produce animmunostimulatory effect.

Method of Stimulating an Anti-Influenza Immune Response

The VLPs of the invention are useful for preparing compositions thatstimulate an immune response that confers immunity or substantialimmunity to influenza viruses. Both mucosal and cellular immunity maycontribute to immunity to influenza infection and disease. Antibodiessecreted locally in the upper respiratory tract are a major factor inresistance to natural infection. Secretory immunoglobulin A (sIgA) isinvolved in protection of the upper respiratory tract and serum IgG inprotection of the lower respiratory tract. The immune response inducedby an infection protects against reinfection with the same virus or anantigenically similar viral strain. Influenza virus undergoes frequentand unpredictable changes; therefore, after natural infection, theeffective period of protection provided by the host's immunity may onlybe a few years against the new strains of virus circulating in thecommunity.

VLPs of the invention can induce substantial immunity in a vertebrate(e.g. a human) when administered to said vertebrate. The substantialimmunity results from an immune response against the influenza VLP ofthe invention that protects or ameleorates influenza infection or atleast reduces a symptom of influenza virus infection in said vertebrate.In some instances, if the said vertebrate is infected, said infectionwill be asymptomatic. The response may be not a fully protectiveresponse. In this case, if said vertebrate is infected with an influenzavirus, the vertebrate will experience reduced symptoms or a shorterduration of symptoms compared to a non-immunized vertebrate.

In one embodiment, the invention comprises a method of inducingsubstantial immunity to influenza virus infection or at least onesymptom thereof in a subject, comprising administering at least oneeffective dose of an influenza VLP. In another embodiment, saidinduction of substantial immunity reduces duration of influenzasymptoms. In another embodiment, a method to induce substantial immunityto influenza virus infection or at least one symptom thereof in asubject, comprises administering at least one effective dose of aninfluenza VLP, wherein said VLP comprises influenza HA, NA and M1proteins. In another embodiment, said influenza VLP comprises influenzaproteins, wherein said influenza proteins consist of HA, NA and M1proteins. These VLPs contain HA, NA and M1 and may contain additionalcellular constituents such as cellular proteins, baculovirus proteins,lipids, carbohydrates etc., but do not contain additional influenzaproteins (other than fragments of M1, HA and/or NA). In anotherembodiment, a method of inducing substantial immunity to influenza virusinfection or at least one symptom thereof in a subject, comprisesadministering at least one effective dose of an influenza VLP, whereinsaid VLP consists essentially of influenza HA, NA and M1. Said VLPs maycomprise additional influenza proteins and/or protein contaminates innegligible concentrations. In another embodiment, a method of inducingsubstantial immunity to influenza virus infection or at least onesymptom thereof in a subject, comprises administering at least oneeffective dose of an influenza VLP, wherein said VLP consists ofinfluenza HA, NA and M1. In another embodiment, said HA and/or NAexhibits hemagglutinin activity and/or neuraminidase activity,respectfully. In another embodiment, said subject is a mammal. Inanother embodiment, said mammal is a human. In a further embodiment,said VLP is formulated with an adjuvant or immune stimulator.

Recently there has been a concerted effort to create a vaccine againstavian influenza virus that has the potential to create a pandemic. Thatis because a number of avian influenza viruses have crossed the speciesbarrier and directly infected humans resulting in illness and, in somecases, death. These viruses were H5N1, H9N2 and H7N7 (Cox et al., 2004).A recent study examined the potential of using inactivated H5N1influenza virus as a vaccine. The formulation of the vaccine was similarto the licensed inactivated vaccines currently licensed for marketing.The study concluded that using inactivated H5N1 virus did induce animmune response in humans, however the dose given was very high (90 μgof avian influenza compared to 15 μg of the licensed vaccine) (Treanoret al., 2006). This high amount of avian influenza antigen isimpractical for a worldwide vaccination campaign. As illustrated below,the VLPs of the invention induces an immune response in a vertebratewhen administered to said vertebrate.

Thus, the invention encompasses a method of inducing substantialimmunity to influenza virus infection or at least one symptom thereof ina subject, comprising administering at least one effective dose of anavian influenza VLP. In another embodiment, said induction ofsubstantial immunity reduces duration of influenza symptoms. In anotherembodiment, said induction of immunity is from administering at least0.2 μg of avian HA in VLPs of the invention. In another embodiment, saidinduction of immunity is from administering about 0.2 μg of avian HA toabout 15 μg of avian HA in VLPs of the invention. Administration may bein one or more doses, but may be advantageously in a single dose. Inanother embodiment, said VLP avian HA is derived from avian influenzaH5N1.

In another embodiment, the invention comprises a method of inducingsubstantial immunity to avian influenza virus infection or at least onesymptom thereof in a subject comprising administering at least oneeffective dose of an avian influenza VLP, wherein said VLP comprises anavian influenza HA, NA and M1. In another embodiment, said avianinfluenza VLP comprises avian influenza proteins, wherein said avianinfluenza proteins consist of HA, NA and M1 proteins. These VLPs containHA, NA and M1 and may contain additional cellular constituents such ascellular proteins, baculovirus proteins, lipids, carbohydrates etc. butdo not contain additional influenza proteins (other than fragments ofM1, HA and/or NA). In another embodiment, said method of inducingsubstantial immunity to avian influenza virus infection or at least onesymptom thereof in a subject comprises administering at least oneeffective dose of an avian influenza VLP, wherein said VLP consistsessentially of avian influenza HA, NA and M1. Said VLPs may compriseadditional influenza proteins and/or protein contaminates in negligibleconcentrations. In another embodiment, a method to induce substantialimmunity to influenza virus infection or at least one symptom thereof ina subject, comprises administering at least one effective dose of aninfluenza VLP, wherein said VLP consists of avian influenza HA, NA andM1. In another embodiment, said avian influenza HA and NA are H5N1,respectively. In another embodiment, said avian influenza HA and NA areH9N2, respectively. In another embodiment, said avian influenza HA andNA are H7N7, respectively. In another embodiment, said avian influenzaHA and/or NA exhibits hemagglutinin activity and/or neuraminidaseactivity, respectfully. In another embodiment, said subject is a mammal.In another embodiment, said mammal is a human. In a further embodiment,said VLP is formulated with an adjuvant or immune stimulator.

In another embodiment, said avian influenza VLPs will induce an immuneresponse in a vertebrate that is about 2 fold, about 4 fold, about 8fold, about 16 fold, about 32 fold about 64 fold, about 128 foldincrease (or higher) more potent than a similar avian influenza antigensformulated similarly to the licensed inactivated vaccines currentlylicensed for marketing. Current formulations comprise whole inactivatedvirus (e.g. formaldehyde treated), split virus (chemically disrupted),and subunit (purified glycoprotein) vaccines. Methods for determiningpotency for a vaccine are known and routine in the art. For example,microneutralization assays and hemagglutination inhibition assays can beperformed to determine potency of an avian VLP vaccine compared to avianinfluenza antigens formulated similar to the licensed inactivatedvaccines currently licensed for marketing. In one embodiment, saidincrease in potency is realized when about 0.2 μg, about 0.4 μg, about0.6 μg about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg,about 5 μg, about 6 μg, about 7 μg, about 9 μg, about 10 μg, about 15μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, 40 μg, about 45μg, about 50 μg, or higher of VLPs and the antigen formulated similarlyto the inactivated vaccines currently licensed for marketing isadministered to a vertebrate (i.e. equivalent amounts of HA and/or NA ina VLP with equivalent amounts of HA and/or NA formulated in similarly tothe licensed inactivated vaccines and/or any other antigen) Amounts canbe measured according to HA content. For example, 1 μg of a VLP of theinvention is about 1 μg of HA in a solution of VLPs comprising HA or maybe measured by weight of VLPs.

Seasonal influenza vaccines are administered to humans every year toreduce the incidence of influenza cases every year. At present, thereare two subtypes of influenza A and influenza B circulating in theUnited States. Current vaccines are, therefore, trivalent to provideprotection against the strains currently circulating. Each year adifferent stain or variation of an influenza viral changes. Thus, formost years a new vaccine composition is manufactured and administered.Inactivated vaccines are produced by propagation of the virus inembryonated hens' eggs. The allantoic fluid is harvested, and the virusis concentrated and purified, then inactivated. Thus, the currentlicensed influenza virus vaccines may contain trace amounts of residualegg proteins and, therefore, should not be administered to persons whohave anaphylactic hypersesitiviety to eggs. In addition, supplies ofeggs must be organized and strains for vaccine production must beselected months in advance of the next influenza season, thus limitingthe flexibility of this approach and often resulting in delays andshortages in production and distribution. In addition, some influenzastrains do not replicate well in embryonated chicken eggs which maylimit the influenza strains which can be grown and formulated intovaccines.

As mentioned above, VLP of the invention do not require eggs forproduction. These VLPs are made via a cell culture system. Thus, theinvention encompasses a method of inducing substantial immunity toinfluenza virus infection or at least one symptom thereof in a subject,comprising administering at least one effective dose of a seasonalinfluenza VLP. A discussed above, seasonal influenza virus refers to theinfluenza viral strains that has been determined to be passing withinthe human population for a given influenza season based on theepidemiological surveys by National Influenza Centers worldwide. Saidstudies and some isolated influenza viruses are sent to one of fourWorld Health Organization (WHO) reference laboratories, one of which islocated at the Centers for Disease Control and Prevention (CDC) inAtlanta, for detailed testing. These laboratories test how wellantibodies made to the current vaccine react to the circulating virusand new flu viruses. This information, along with information about fluactivity, is summarized and presented to an advisory committee of theU.S. Food and Drug Administration (FDA) and at a WHO meeting. Thesemeetings result in the selection of three viruses (two subtypes ofinfluenza A viruses and one influenza B virus) to go into flu vaccinesfor the following fall and winter. The selection occurs in February forthe northern hemisphere and in September for the southern hemisphere.Usually, one or two of the three virus strains in the vaccine changeseach year. In another embodiment, said induction of substantial immunityreduces duration of influenza symptoms.

In another embodiment, the invention comprises a method of inducingsubstantial immunity to a seasonal influenza virus infection or at leastone symptom thereof in a subject comprising administering at least oneeffective dose of a seasonal influenza VLP, wherein said VLP comprises aseasonal influenza HA, NA and M1. In another embodiment, said seasonalinfluenza VLP comprises seasonal influenza proteins, wherein saidinfluenza proteins consist of HA, NA and M1 proteins. These VLPs containHA, NA and M1 and may contain additional cellular constituents such ascellular proteins, baculovirus proteins, lipids, carbohydrates etc. butdo not contain additional influenza proteins (other than fragments ofM1, HA and/or NA). In another embodiment, said method of inducingsubstantial immunity to seasonal influenza virus infection or at leastone symptom thereof in a subject comprises administering at least oneeffective dose of a seasonal influenza VLP, wherein said VLP consistsessentially of seasonal influenza HA, NA and M1. Said VLPs may compriseadditional influenza proteins and/or protein contaminates in negligibleconcentrations. In another embodiment, a method to induce substantialimmunity to influenza virus infection or at least one symptom thereof ina subject, comprises administering at least one effective dose of aninfluenza VLP, wherein said VLP consists of seasonal influenza HA, NAand M1. In another embodiment, said avian influenza HA and/or NAexhibits hemagglutinin activity and/or neuraminidase activity,respectfully. In another embodiment, said subject is a mammal. Inanother embodiment, said mammal is a human. In a further embodiment,said VLP is formulated with an adjuvant or immune stimulator.

Generally, seasonal influenza VLPs of the invention are administered ina quantity sufficient to stimulate substantial immunity for one or morestrains of seasonal influenza virus. In one embodiment, the VLPs areblended together with other VLPs comprising different influenza subtypesproteins (as listed above). In another embodiment, the formulation is atrivalent formulation which comprises a mixture of VLPs with seasonalinfluenza HA and/or NA proteins from at least two influenza A and/or oneat least one B subtype. In another embodiment, said B subtype isproduced by the same method as described above. In another embodiment, amultivalent formulation comprises one or more of the VLP of theinvention as described above.

In another embodiment, VLPs of the invention (avian or seasonal VLPs)may elicit an immune response that will provide protection against morethan one strain of influenza virus. This cross-protection of avertebrate with an influenza VLP constructed from a particular strain,of a particular subgroup, may induce cross-protection against influenzavirus of different strains and/or subgroups. The examples below showthat VLPs of the invention are capable of inducing cross reactivity withdifferent strains and/or subgroups.

The humoral immune system produces antibodies against differentinfluenza antigens, of which the HA-specific antibody is the mostimportant for neutralization of the virus and thus prevention ofillness. The NA-specific antibodies are less effective in preventinginfection, but they lessen the release of virus from infected cells. Themucosal tissues are the main portal entry of many pathogens, includinginfluenza, and the mucosal immune system provides the first line ofdefense against infection apart from innate immunity. SIgA and, to someextent, IgM are the major neutralizing antibodies directed againstmucosal pathogens preventing pathogen entry and can functionintracellularly to inhibit replication of virus. Nasal secretionscontain neutralizing antibodies particularly to influenza HA and NA,which are primarily of the IgA isotype and are produced locally. Duringprimary infection, all three major Ig classes (IgG, IgA and IgM)specific to HA can be detected by enzyme-linked immunosorbent assay innasal washings, although IgA and IgM are more frequently detected thanIgG. Both IgA and, to some extent, IgM are actively secreted locally,whereas IgG is derived as a serum secretion. In subjects who have alocal IgA response, a serum IgA response also is observed. The local IgAresponse stimulated by natural infection lasts for at least 3-5 months,and influenza-specific, IgA-committed memory cells can be detectedlocally. IgA also is the predominant Ig isotype in local secretionsafter secondary infection, and an IgA response is detected in the serumupon subsequent infection. The presence of locally produced neutralizingantibodies induced by live virus vaccine correlates with resistance toinfection and illness after challenge with wild-type virus.

Resistance to influenza infection or illness is correlated with thelevel of local and/or serum antibody to HA and NA. Serum anti-HAantibodies are the most commonly measured correlate of protectionagainst influenza (Cox et al., 1999). A protective serum antibody(haemagglutination inhibition (HI) titer≧40) response can be detected inapproximately 80% of subjects after natural influenza infection. B cellsproducing all three major Ig classes are present in the peripheral bloodin normal subjects (Cox et al., 1994) and individuals undergoinginfluenza infection. In humans, serum antibodies play a role in bothresistance to and recovery from influenza infection. The level of serumantibody to HA and NA in humans can be correlated with resistance toillness following experimental infection and natural infection. Duringprimary infection, the three major Ig classes can be detected within10-14 days. IgA and IgM levels peak after 2 weeks and then begin todecline, whereas the level of IgG peaks at 4-6 weeks. Whereas IgG andIgM are dominant in the primary response, IgG and IgA predominate in thesecondary immune response.

Thus, the invention encompasses a method of inducing a substantiallyprotective antibody response to influenza virus infection or at leastone symptom thereof in a subject, comprising administering at least oneeffective dose of an influenza VLP. In another embodiment, saidinduction of substantially protective antibody response reduces durationof influenza symptoms. In another embodiment, a method to inducesubstantially protective antibody response to influenza virus infectionor at least one symptom thereof in a subject, comprises administering atleast one effective dose of an influenza VLP, wherein said VLP comprisesinfluenza HA, NA and M1 proteins.

In another embodiment, the invention comprises a method of inducingsubstantially protective antibody response to influenza virus infectionor at least one symptom thereof in a subject, comprises administering atleast one effective dose of an influenza VLP, wherein said VLP consistsessentially of influenza HA, NA and M1. Said VLPs may compriseadditional influenza proteins and/or protein contaminates in negligibleconcentrations. In another embodiment, said influenza VLP comprisesinfluenza proteins, wherein said influenza proteins consist of HA, NAand M1 proteins. These VLPs contain HA, NA and M1 and may containadditional cellular constituents such as cellular proteins, baculovirusproteins, lipids, carbohydrates etc., but do not contain additionalinfluenza proteins (other than fragments of M1, HA and/or NA). Inanother embodiment, a method of inducing substantial immunity toinfluenza virus infection or at least one symptom thereof in a subject,comprises administering at least one effective dose of an influenza VLP,wherein said VLP consists of influenza HA, NA and M1. In anotherembodiment, wherein said influenza HA, NA and M1 is derived fromseasonal influenza and/or avian influenza. In another embodiment, saidHA and/or NA exhibits hemagglutinin activity and/or neuraminidaseactivity, respectfully. In another embodiment, said subject is a mammal.In another embodiment, said mammal is a human. In a further embodiment,said VLP is formulated with an adjuvant or immune stimulator.

As used herein, an “antibody” is a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes. The recognized immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. A typical immunoglobulin (antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kD) andone “heavy” chain (about 50-70 kD). The N-terminus of each chain definesa variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. Antibodies exist as intactimmunoglobulins or as a number of well-characterized fragments producedby digestion with various peptidases.

Cell-mediated immunity also plays a role in recovery from influenzainfection and may prevent influenza-associated complications.Influenza-specific cellular lymphocytes have been detected in the bloodand the lower respiratory tract secretions of infected subjects.Cytolysis of influenza-infected cells is mediated by CTLs in concertwith influenza-specific antibodies and complement. The primary cytotoxicresponse is detectable in blood after 6-14 days and disappears by day 21in infected or vaccinated individuals (Ennis et al., 1981).Influenza-specific CTLs exhibit cross-reactive specificities in in vitrocultures; thus, they lyse cells infected with the same type of influenzabut not with other types (e.g. influenza A but not influenza B virus).CTLs that recognize the internal nonglycosylated proteins, M, NP and PB2have been isolated (Fleischer et al., 1985). The CTL response iscross-reactive between influenza A strains (Gerhard et al., 2001) and isimportant in minimizing viral spread in combination with antibody(Nguyen et al., 2001).

Cell-mediated immunity also plays a role in recovery from influenzainfection and may prevent influenza-associated complications.Influenza-specific cellular lymphocytes have been detected in the bloodand the lower respiratory tract secretions of infected subjects.Cytolysis of influenza-infected cells is mediated by CTLs in concertwith influenza-specific antibodies and complement. The primary cytotoxicresponse is detectable in blood after 6-14 days and disappears by day 21in infected or vaccinated individuals (Ennis et al., 1981).Influenza-specific CTLs exhibit cross-reactive specificities in in vitrocultures; thus, they lyse cells infected with the same type of influenzabut not with other types (e.g. influenza A but not influenza B virus).CTLs that recognize the internal nonglycosylated proteins, M, NP and PB2have been isolated (Fleischer et al., (1985). The CTL response iscross-reactive between influenza A strains (Gerhard et al., 2001) and isimportant in minimizing viral spread in combination with antibody(Nguyen et al., 2001).

Thus, the invention encompasses a method of inducing a substantiallyprotective cellular immune response to influenza virus infection or atleast one symptom thereof in a subject, comprising administering atleast one effective dose of an influenza VLP. In another embodiment, amethod of inducing substantial immunity to influenza virus infection orat least one symptom thereof in a subject, comprises administering atleast one effective dose of an influenza VLP, wherein said VLP consistsof influenza HA, NA and M1. In another embodiment, said influenza VLPcomprises influenza proteins, wherein said influenza proteins consist ofHA, NA and M1 proteins. These VLPs contain HA, NA and M1 and may containadditional cellular constituents such as cellular proteins, baculovirusproteins, lipids, carbohydrates etc. but do not contain additionalinfluenza proteins (other than fragments of M1, HA and/or NA). Inanother embodiment wherein said influenza HA, NA and M1 is derived fromseasonal influenza and/or avian influenza virus. In another embodiment,said HA and/or NA exhibits hemagglutinin activity and/or neuraminidaseactivity, respectfully. In another embodiment, said subject is a mammal.In another embodiment, said mammal is a human. In a further embodiment,said VLP is formulated with an adjuvant or immune stimulator.

As mentioned above, the VLPs of the invention (e.g. avian and/orseasonal influenza VLPs) prevent or reduce at least one symptom ofinfluenza infection in a subject. Symptoms of influenza are well knownin the art. They include fever, myalgia, headache, severe malaise,nonproductive cough, sore throat, weight loss and rhinitis. Thus, themethod of the invention comprises the prevention or reduction of atleast one symptom associated with influenza viral infection. A reductionin a symptom may be determined subjectively or objectively, e.g., selfassessment by a subject, by a clinician's assessment or by conducting anappropriate assay or measurement (e.g. body temperature), including,e.g., a quality of life assessment, a slowed progression of an influenzainfection or additional symptoms, a reduced severity of a influenzasymptoms or a suitable assays (e.g. antibody titer and/or T-cellactivation assay). The objective assessment comprises both animal andhuman assessments.

The principal strategy advocated by the Advisory Committee onImmunization Practices (ACIP) for control of influenza has been thevaccination of persons at risk for serious complications from influenza,in particular, people≧65 years old. Yearly influenza epidemics, however,continue unabated and are responsible for significant health andfinancial burden to our society (Glaser et al., 1996). In the last 20years (1976-1999), a significant increase has occurred ininfluenza-associated all cause excess deaths. From 1990 to 1999, theannual number of influenza-associated all cause deaths exceeded 50,000(Thompson et al., 2003). Despite the increase in vaccine coverage ofpeople≧65 years to 65% during the last decade, a corresponding reductionin influenza-associated all cause excess deaths has not been observed.

Thus, another strategy for the prevention and control of influenza isuniversal vaccination of healthy children and individuals. Children havehigh rates of infection, medically attended illness and hospitalizationfrom influenza (Neuzil et al., 2000). Children play an important role inthe transmission of influenza within schools, families and communities.Vaccination with current influenza vaccines of approximately 80% ofschoolchildren in a community has decreased respiratory illnesses inadults and excess deaths in the elderly (Reichert et al., 2001). Thisconcept is known as community immunity or “herd immunity” and is thoughtto play an important part of protecting the community against disease.Because vaccinated people have antibodies that neutralize influenzavirus, they are much less likely to transmit influenza virus to otherpeople. Thus, even people who have not been vaccinated (and those whosevaccinations have become weakened or whose vaccines are not fullyeffective) often can be shielded by the herd immunity because vaccinatedpeople around them are not getting sick. Herd immunity is more effectiveas the percentage of people vaccinated increases. It is thought thatapproximately 95% of the people in the community must be protected by avaccine to achieve herd immunity. People who are not immunized increasethe chance that they and others will get the disease.

Thus, the invention encompasses a method of inducing a substantiallyprotective immunity to influenza virus infection to a population or acommunity in order to reduce the incidence of influenza virus infectionsamong immunocompromised individuals or non-vaccinated individual buyadministering VLPs of the invention to a population in a community. Inone embodiment, most school-aged children are immunized againstinfluenza virus by administering the VLPs of the invention. In anotherembodiment, most healthy individuals in a community to are immunizedagainst influenza virus by administering the VLPs of the invention. Inanother embodiment VLPs of the invention are part of a “dynamicvaccination” strategy. Dynamic vaccination is the steady production of alow-efficacy vaccine that is related to an emerging pandemic strain, butdue to an antigentic drift may not provide complete protection in amammal (see Germann et al., 2006). Because of the uncertainty about thefuture identity of a pandemic strain, it is almost impossible tostockpile a well matched pandemic strain. However, vaccination with apoorly matched but potentially efficacious vaccine may slow the spreadof the pandemic virus and/or reduce the severity of symptoms of apandemic strain of influenza virus.

The invention also encompasses a vaccine comprising an influenza VLP,wherein said vaccine induces substantial immunity to influenza virusinfection or at least one symptom thereof when administered to asubject. In another embodiment, said induction of substantial immunityreduces duration of influenza symptoms. In another embodiment, a saidvaccine induces substantial immunity to influenza virus infection or atleast one symptom thereof in a subject, comprises a VLP which comprisesinfluenza HA, NA and M1 proteins. In another embodiment, a said vaccineinduces substantial immunity to influenza virus infection or at leastone symptom thereof in a subject, comprises a VLP which consistsessentially of influenza HA, NA and M1 proteins. Said VLPs may compriseadditional influenza proteins and/or protein contaminates in negligibleconcentrations. In another embodiment, a said vaccine inducessubstantial immunity to influenza virus infection or at least onesymptom thereof in a subject, comprises a VLP which consists ofinfluenza HA, NA and M1 proteins. In another embodiment, a said vaccineinduces substantial immunity to influenza virus infection or at leastone symptom thereof in a subject, comprises a VLP comprises influenzaproteins, wherein said influenza proteins consist of HA, NA and M1proteins. These VLPs contain HA, NA and M1 and may contain additionalcellular constituents such as cellular proteins, baculovirus proteins,lipids, carbohydrates etc., but do not contain additional influenzaproteins (other than fragments of M1, HA and/or NA). In anotherembodiment, said influenza HA, NA and M1 proteins are derived from anavian and/or seasonal influenza virus. In another embodiment, said HAand/or NA exhibits hemagglutinin activity and/or neuraminidase activity,respectfully. In another embodiment, said subject is a mammal. Inanother embodiment, said mammal is a human. In a further embodiment,said VLP is formulated with an adjuvant or immune stimulator. In anotherembodiment, where said vaccine is administered to a mammal. In a furtherembodiment, said mammal is a human.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing, areincorporated herein by reference.

EXAMPLES Example 1 Materials and Methods

Avian influenza A/Hong Kong/1073/99 (H9N2) virus HA, NA, and M1 geneswere expressed in Spodoptera frugiperda cells (Sf-9S cell line; ATCCPTA-4047) using the baculovirus bacmid expression system. The HA, NA,and M1 genes were synthesized by the reverse transcription andpolymerase chain reaction (PCR) using RNA isolated from avian influenzaA/Hong Kong/1073/99 (H9N2) virus (FIGS. 1, 2, and 3). For reversetranscription and PCR, oligonucleotide primers specific for avianinfluenza A/Hong Kong/1073/99 (H9N2) virus HA, NA, and M1 genes wereused (Table 1). The cDNA copies of these genes were cloned initiallyinto the bacterial subcloning vector, pCR2.1TOPO. From the resultingthree pCR2.1TOPO-based plasmids, the HA, NA, and M1 genes were inserteddownstream of the AcMNPV polyhedrin promoters in the baculovirustransfer vector, pFastBac1 (InVitrogen), resulting in threepFastBac1-based plasmids: pHA, pNA, and pM1 expressing these influenzavirus genes, respectively. Then, a single pFastBac1-based plasmid pHAMwas constructed encoding both the HA and M1 genes, each downstream froma separate polyhedrin promoter (FIG. 4). The nucleotide sequence of theNA gene with the adjacent 5′- and 3′-regions within the pNA plasmid wasdetermined (SEQ ID NO:1) (FIG. 1). At the same time, the nucleotidesequences of the HA and M1 genes with the adjacent regions were alsodetermined using the pHAM plasmid (SEQ ID NOS:2 and 3) (FIGS. 2 and 3).

Finally, a restriction DNA fragment from the pHAM plasmid that encodedboth the HA and M1 expression cassettes was cloned into the pNA plasmid.This resulted in the plasmid pNAHAM encoding avian influenza A/HongKong/1073/99 (H9N2) virus HA, NA, and M1 genes (FIG. 4).

Plasmid pNAHAM was used to construct a recombinant baculoviruscontaining influenza virus NA, HA, and M1 genes integrated into thegenome, each downstream from a separate baculovirus polyhedrin promoter.Infection of permissive Sf-9S insect cells with the resultingrecombinant baculovirus resulted in co-expression of these threeinfluenza genes in each Sf-9S cell infected with such recombinantbaculovirus.

The expression products in infected Sf-9S cells were characterized at 72hr postinfection (p.i. by SDS-PAGE analysis, Coomassie blue proteinstaining, and Western immunoblot analysis using HA- and M1-specificantibodies (FIG. 5). Western immunoblot analysis was carried out usingrabbit antibody raised against influenza virus type A/Hong Kong/1073/99(H9N2) (CDC, Atlanta, Ga., USA), or mouse monoclonal antibody toinfluenza M1 protein (Serotec, UK). The HA, NA, and M1 proteins of theexpected molecular weights (64 kd, 60 kd, and 31 kd, respectively) weredetected by Western immunoblot analysis. Compared to the amount of HAprotein detected in this assay, the NA protein showed lower reactivitywith rabbit serum to influenza A/Hong Kong/1073/99 (H9N2) virus.Explanations for the amount of detectable NA protein included lowerexpression levels of the NA protein from Sf-9S cells infected withrecombinant baculovirus as compared to the HA protein, lower reactivityof the NA with this serum under denaturing conditions in the Westernimmunoblot assay (due to the elimination of important NA epitopes duringgel electrophoresis of membrane binding), lower NA-antibody avidity ascompared to HA-antibody, or a lower abundance of NA-antibodies in theserum.

The culture medium from the Sf-9S cells infected with recombinantbaculovirus expressing A/Hong Kong/1073/99 (H9N2) HA, NA, and M1proteins was also probed for influenza proteins. The clarified culturesupernatants were subjected to ultracentrifugation at 27,000 rpm inorder to concentrate high-molecular protein complexes of influenzavirus, such as subviral particles, VLP, complexes of VLP, and possibly,other self-assembled particulates comprised of influenza HA, NA, and M1proteins. Pelleted protein products were resuspended inphosphate-buffered saline (PBS, pH 7.2) and further purified byultracentrifugation on discontinuous 20-60% sucrose step gradients.Fractions from the sucrose gradients were collected and analyzed bySDS-PAGE analysis, Western immunoblot analysis, and electron microscopy.

Influenza HA and M1 proteins of the expected molecular weights weredetected in multiple sucrose density gradient fractions by Coomassieblue staining and Western immunoblot analysis (FIG. 6, Table 1). Thissuggested that influenza viral proteins from infected Sf-9S cells areaggregated in complexes of high-molecular weight, such as capsomers,subviral particles, VLP, and/or VLP complexes. The NA proteins, althoughinconsistently detected by Coomassie blue staining and Westernimmunoblot analysis, which was likely due to the inability of the rabbitanti-influenza serum to recognize denatured NA protein in the Westernimmunoblot assay, were consistently detected in neuraminidase enzymeactivity assay (FIG. 10).

TABLE 1 Fraction#* Titer 1 <1:5001 3 <1:500 5   1:500 7   1:1000 9  1:2000 11   1:2000 12   1:4000 14   1:500 16 <1:500 PBS** <1:500A/Shangdong/9/93 <1:1000 *Fraction from 20-60% sucrose gradient**Negative Control ***Positive Control

SEQ ID Virus Strain Gene RT-PCR Primer NO Type A (H3N2) HemagglutininForward 5′-A GGATCC ATG AAGACTATCATTGCTTTGAG-3′ 13 Sydney/ (HA) Reverse5′-A GGTACC TCAAATGCAAATGTTGCACCTAATG-3′ 14 5/97 Neuraminidase Forward5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGAAG 15 (NA)GAGATAGAACC ATG AATCCAAATCAAAAGATAATAAC-3′ Reverse5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATAT 16 AGGCATGAGATTGATGTCCGC-3′Matrix (M1) Forward 5′-AAA GAATTC ATG AGTCTTCTAACCGAGGTCGAAACGTA-3′ 17Reverse 5′-AAA TTCGAA TTACTCCAGCTCTATGCTGACAAAATGAC-3′ 18 M2 Forward5′-A GAATC ATG AGTCTTCTAACCGAGGTCGAAACGCCT 19 ATCAGAAACGAATGGGGGTGC-3′Reverse 5′-AAA TTCGAA TTACTCCAGCTCTATGCTGACAAAATGAC-3′ 20 NucleoproteinForward 5′-A GAATTC ATG GCGTCCCAAGGCACCAAACG-3′ 21 (NP) Reverse5′-AGCGGCCGCTTAATTGTCGTACTCCTCTGCATTGTCTCCGAA 22 GAAATAAG-3′ Type BHarbin Hemagglutinin Forward5′-A GAATTC ATG AAGGCAATAATTGTACTACTCATGG-3′ 23 (HA) Reverse5′-A GCGGCCGCTTATAGACAGATGGAGCAAGAAACATTGTC 24 TCTGGAGA-3′ NeuraminidaseForward 5′-A GAATT C ATG CTACCTTCAACTATACAAACG-3′ 25 (NA) Reverse5′-AGCGGCCGCTTACAGAGCCATATCAACACCTGTGACAGTG-3′ 26

The presence of high-molecular VLPs was confirmed by gel filtrationchromatography. An aliquot from sucrose density gradient fractionscontaining influenza viral proteins was loaded onto a Sepharose CL-4Bcolumn for fractionation based on mass. The column was calibrated withdextran blue 2000, dextran yellow, and vitamin B12 (Amersham Pharmacia)with apparent molecular weights of 2,000,000; 20,000; and 1,357 daltons,respectively, and the void volume of the column was determined. Asexpected, high-molecular influenza viral proteins migrated in the voidvolume of the column, which was characteristic of macromolecularproteins, such as virus particles. Fractions were analyzed by Westernimmunoblot analysis to detect influenza and baculovirus proteins. Forexample, M1 proteins were detected in the void volume fractions, whichalso contained baculovirus proteins (FIG. 7).

The morphology of influenza VLPs and proteins in sucrose gradientfractions was elucidated by electron microscopy. For negative-stainingelectron microscopy, influenza proteins from two sucrose densitygradient fractions were fixed with 2% glutaraldehyde in PBS, pH 7.2.Electron microscopic examination of negatively-stained samples revealedthe presence of macromolecular protein complexes or VLPs in bothfractions. These VLPs displayed different sizes including diameters ofapproximately 60 and 80 nm and morphologies (spheres). Larger complexesof both types of particles were also detected, as well as rod-shapedparticles (FIG. 8). All observed macromolecular structures had spikes(peplomers) on their surfaces, which is characteristic of influenzaviruses. Since the size and appearance of 80 nm particles was similar toparticles of wild type influenza virus, these structures likelyrepresented VLPs, which have distinct similarities to wild typeinfluenza virions, including similar particle geometry, architecture,triangulation number, symmetry, and other characteristics. The smallerparticles of approximately 60 nm may represent subviral particles thatdiffer from VLPs both morphologically and structurally. Similarphenomenon of recombinant macromolecular proteins of different sizes andmorphologies was also reported for other viruses. For example,recombinant core antigen (HBcAg) of hepatitis B virus forms particles ofdifferent sizes, which have different architecture and triangulationnumber T=4 and T=3, respectively (Crowther et al., 1994).

To characterize the functional properties of the purified influenzaA/Hong Kong/1073/99 (H9N2) VLPs, samples were tested in ahemagglutination assay (FIG. 9) and a neuraminidase enzyme assay (FIG.10). For the hemagglutination assay, 2-fold dilutions of purifiedinfluenza VLPs were mixed with 0.6% guinea pig red blood cells andincubated at 4° C. for 1 hr or 16 hr. The extent of hemagglutination wasinspected visually and the highest dilution of recombinant influenzaproteins capable of agglutinating red blood cells was determined andrecorded (FIG. 9). Again, many fractions from the sucrose densitygradient exhibited hemagglutination activity, suggesting that multiplemacromolecular and monomeric forms of influenza proteins were present.The highest titer detected was 1:4000. In a control experiment,wild-type influenza A/Shangdong virus demonstrated a titer of 1:2000.The hemagglutination assay revealed that the recombinant VLPs consistingof influenza A/Hong Kong/1073/99 (H9N2) virus HA, NA, and M1 proteinswere functionally active. This suggested that the assembly,conformation, and folding of the HA subunit proteins within the VLPswere similar or identical to that of the wild type influenza virus.

Additionally, a neuraminidase enzyme assay was performed on samples ofpurified H9N2 VLPs. The amount of neuraminidase activity in sucrosedensity gradient fractions was determined using fetuin as a substrate.In the neuraminidase assay, the neuraminidase cleaved sialic acid fromsubstrate molecules to release sialic acid for measurement. Arsenitereagent was added to stop enzyme activity. The amount of sialic acidliberated was determined chemically with thiobarbituric acid thatproduces a pink color that was proportional to the amount of free sialicacid. The amount of color (chromophor) was measuredspectrophotometrically at wavelength 549 nm. Using this method,neuraminidase activity was demonstrated in sucrose gradient fractionscontaining influenza VLPs (FIG. 10). As expected, the activity wasobserved in several fractions, with two peak fractions. As a positivecontrol, wild type influenza virus was used. The wild type influenzavirus exhibited neuraminidase enzyme activity comparable to that ofpurified influenza VLPs. These findings corroborated the HA results withregard to protein conformation and suggested that purified VLPs ofinfluenza A/Hong Kong/1073/99 (H9N2) virus were functionally similar towild type influenza virus.

The results from the above analyses and assays indicated that expressionof influenza A/Hong Kong/1073/99 (H9N2) HA, NA, and M1 proteins wassufficient for the self-assembly and transport of functional VLPs frombaculovirus-infected insect cells. Since these influenza VLPsrepresented self-assembled influenza structural proteins anddemonstrated functional and biochemical properties similar to those ofwild type influenza virus, these influenza VLPs conserved importantstructural conformations including surface epitopes necessary foreffective influenza vaccines.

Example 2 RT-PCR Cloning of Avian Influenza A/Hong Kong/1073/99 ViralGenes

It is an object of the present invention to provide synthetic nucleicacid sequences capable of directing production of recombinant influenzavirus proteins. Such synthetic nucleic acid sequences were obtained byreverse transcription and polymerase chain reaction (PCR) methods usinginfluenza virus natural genomic RNA isolated from the virus. For thepurpose of this application, nucleic acid sequence refers to RNA, DNA,cDNA or any synthetic variant thereof which encodes the protein.

Avian influenza A/Hong Kong/1073/99 (H9N2) virus was provided by Dr. K.Subbarao (Centers for Disease Control, Atlanta, Ga., USA). Viral genomicRNA was isolated by the acid phenol RNA extraction method underBiosafety Level 3 (BSL3) containment conditions at CDC using Trizol LSreagent (Invitrogen, Carlsbad, Calif. USA). cDNA molecules of the viralRNAs were obtained by reverse transcription using MuLV reversetranscriptase (InVitrogen) and PCR using oligonucleotide primersspecific for HA, NA, and M1 proteins and Taq I DNA polymerase(InVitrogen) (Table 1). The PCR fragments were cloned into the bacterialsubcloning vector, pCR2.1TOPO (InVitrogen), between Eco RI sites thatresulted in three recombinant plasmids, containing the HA, NA, and M1cDNA clones.

Example 3 RT-PCR Cloning of Human Influenza A/Sydney/5/94 (H3N2) ViralGenes

Influenza A/Sydney/5/97 (H3N2) Virus was obtained from Dr. M. Massare(Novavax, Inc., Rockville, Md.). Viral genomic RNA was isolated by theRNA acid phenol extraction method under BSL2 containment conditions atNovavax, Inc. using Trizol LS reagent (Invitrogen). cDNA molecules ofthe viral RNAs were obtained by reverse transcription and PCR usingoligonucleotide primers specific for HA, NA, M1, M2, and NP proteins(Table 1). The PCR fragments were cloned into the bacterial subcloningvector, pCR2.1 TOPO, between Eco RI sites that resulted in fiverecombinant plasmids, containing the HA, NA, M1, M2, and NP cDNA clones.

Example 4 Cloning of Avian Influenza A/Hong Kong/1073/99 Viral cDNAsinto Baculovirus Transfer Vectors

From the pCR2.1TOPO-based plasmids, the HA, NA, or M1 genes weresubcloned into pFastBac1 baculovirus transfer vector (InVitrogen) withinthe polyhedron locus and Tn7 att sites and downstream of the baculoviruspolyhedrin promoter and upstream of the polyadenylation signal sequence.The viral genes were ligated with T4 DNA ligase. For the HA gene, a BamHI-Kpn I DNA fragment from pCR2.1TOPO-HA was inserted into BamHI-KpnIdigested pFastBac1 plasmid DNA. For the NA gene, an EcoRI DNA fragmentfrom pCR2.1TOPO-NA was inserted into EcoRI digested pFastBac1 plasmidDNA. For the M1 gene, an Eco RIDNA fragment from pCR2.1TOPO-M1 wasinserted into Eco RI digested pFastBac1 plasmid DNA. Competent E. coliDH5α bacteria (InVitrogen) were transformed with these DNA ligationreactions, transformed colonies resulted, and bacterial clones isolated.The resulting pFastBac1-based plasmids, pFastBac1-HA, pFastBac1-NA, andpFastBac1-M1 were characterized by restriction enzyme mapping on agarosegels (FIG. 4A). The nucleotide sequences as shown on FIGS. 1-3 of thecloned genes were determined by automated DNA sequencing. DNA sequenceanalysis showed that the cloned influenza HA, NA, and M1 genes wereidentical to the nucleotide sequences for these genes as publishedpreviously [NA, HA, and M1 genes of influenza A/Hong Kong/1073/99 (H9N2)(GenBank accession numbers AJ404629, AJ404626, and AJ278646,respectively)].

Example 5 Cloning of Human Influenza A/Sydney/5/97 Viral cDNAs intoBaculovirus Transfer Vectors

From the pCR2.1TOPO-based plasmids, the HA, NA, M1, M2; and NP geneswere subcloned into pFastBac1 baculovirus transfer vector within thepolyhedron locus and Tn7 att sites and downstream of the baculoviruspolyhedrin promoter and upstream of the polyadenylation signal sequence.The viral genes were ligated with T4 DNA ligase. For the HA gene, a BamHI-Kpn I DNA fragment from pCR2.1TOPO-hHA3 was inserted into BamHI-KpnIdigested pFastBac1 plasmid DNA. For the NA gene, an Eco RI DNA fragmentfrom pCR2.1TOPO-hNA was inserted into EcoRI digested pFastBac1 plasmidDNA. For the M1 gene, an Eco RI DNA fragment from pCR2.1TOPO-hM1 wasinserted into EcoRI digested pFastBac1 plasmid DNA. For the M2 gene, anEcoRI DNA fragment from pCR2.1TOPO-hM2 was inserted into EcoRI digestedpFastBac1 plasmid DNA. For the NP gene, an EcoRI DNA fragment frompCR2.1TOPO-hNP was inserted into EcoRI digested pFastBac1 plasmid DNA.Competent E. coli DH5α bacteria were transformed with these DNA ligationreactions, transformed colonies resulted, and bacterial clones isolated.The resulting pFastBac 1-based plasmids, pFastBac1-hHA3, pFastBac1-hNA2,pFastBac1-hM1, pFASTBAC1-hM2, and pFASTBAC1-hNP were characterized byrestriction enzyme mapping on agarose gels. The nucleotide sequences ofthe cloned genes were determined by automated DNA sequencing. DNAsequence analysis showed that the cloned influenza HA, NA, M1, M2, andNP genes were identical to the nucleotide sequences for these genes aspublished previously.

Example 6 Construction of Multigenic Baculovirus Transfer VectorsEncoding Multiple Avian Influenza A/Hong Kong/1073/99 Viral Genes

In order to construct pFastBac1-based bacmid transfer vectors expressingmultiple influenza A/Hong Kong/1073/99 (H9N2) virus genes, initially aSna BI-Hpa I DNA fragment from pFastBac1-M1 plasmid containing the M1gene was cloned into Hpa I site of pFastBac1-HA. This resulted inpFastBac1-HAM plasmid encoding both HA and M1 genes within independentexpression cassettes and expressed under the control of separatepolyhedrin promoters.

Finally, a SnaBI-AvrII DNA fragment from pFastBac1-HAM containing the HAand M1 expression cassettes, was transferred into Hpa I-Avr II digestedpFastBac1-NA plasmid DNA. This resulted in the plasmid pFastBac1-NAHAMencoding three independent expression cassettes for expression ofinfluenza HA, NA, and M1 genes and expressed under the control ofseparate polyhedrin promoters (FIG. 4B).

In another example, the H3 gene from pFASTBAC1-hHA3 (see Example 5) wascloned into pFASTBAC1-NAHAM as a fourth influenza viral gene for theexpression and production of heterotypic influenza VLPs.

Example 7 Generation of Multigenic Recombinant Baculovirus Encoding NA,HA, and M1 Genes of Avian Influenza A/Hong Kong/1073/99 Virus in InsectCells

The resulting multigenic bacmid transfer vector pFastBac1-NAHAM was usedto generate a multigenic recombinant baculovirus encoding avianinfluenza A/Hong Kong/1073/99 (H9N2) HA, NA, and M1 genes for expressionin insect cells. Recombinant bacmid DNAs were produced by site-specificrecombination at polyhedrin and Tn7 att DNA sequences betweenpFastBac1-NAHAM DNA and the AcMNPC baculovirus genome harbored incompetent E. coli DH10BAC cells (InVitrogen) (FIG. 4B). Recombinantbacmid DNA was isolated by the mini-prep plasmid DNA method andtransfected into Sf-9s cells using the cationic lipid CELLFECTIN(InVitrogen). Following transfection, recombinant baculoviruses wereisolated, plaque purified, and amplified in Sf-9S insect cells. Virusstocks were prepared in Sf-9S insect cells and characterized forexpression of avian influenza viral HA, NA, and M1 gene products. Theresulting recombinant baculovirus was designated bNAHAM-H9N2.

Example 8 Expression of Recombinant Avian Influenza A/Hong Kong/1073/99Proteins in Insect Cells

Sf-9S insect cells maintained as suspension cultures in shaker flasks at28° C. in serum-free medium (HyQ SFM, HyClone, Ogden, Utah) wereinfected at a cell density of 2×10⁶ cells/ml with the recombinantbaculovirus, bNAHAM-H9N2, at a multiplicity of infection (MOI) of 3pfu/cell. The virus infection proceeded for 72 hrs. to allow expressionof influenza proteins. Expression of avian influenza A/Hong Kong/1073/99(H9N2) HA and M1 proteins in infected insect cells was confirmed bySDS-PAGE and Western immunoblot analyses. SDS-PAGE analysis wasperformed on 4-12% linear gradient NuPAGE gels (Invitrogen) underreduced and denaturing conditions. Primary antibodies in Westernimmunoblot analysis were polyclonal rabbit antiserum raised againstavian influenza A/Hong Kong/1073/99 (H9N2) obtained from CDC andmonoclonal murine antiserum to influenza M1 protein (Serotec, UK).Secondary antibodies for Western immunoblot analysis were alkalinephosphatase conjugated goat IgG antisera raised against rabbit or mouseIgG (H+ L) (Kirkegaard and Perry Laboratories, Gaithersburg, Md., USA).Results of these analyses (FIG. 5) indicated that the HA and M1 proteinswere expressed in the baculovirus-infected insect cells.

Example 9 Purification of Recombinant Avian Influenza H9N2 Virus-LikeParticles and Macromolecular Protein Complexes

Culture supernatants (200 ml) from Sf-9S insect cells infected with therecombinant baculovirus bNAHAM-H9N2 that expressed avian influenzaA/Hong Kong/1073/99 (H9N2) HA, NA, and M1 gene products were harvestedby low speed centrifugation. Culture supernatants were clarified bycentrifugation in a Sorval RC-5B superspeed centrifuge for 1 hr at10,000×g and 4° C. using a GS-3 rotor. Virus and VLPs were isolated fromclarified culture supernatants by centrifugation in a Sorval OTD-65ultracentrifuge for 3 hr at 27,000 rpm and 4° C. using a Sorval TH-641swinging bucket rotor. The virus pellet was resuspended in 1 ml of PBS(pH 7.2), loaded onto a 20-60% (w/v) discontinuous sucrose stepgradient, and resolved by centrifugation in a Sorval OTD-65ultracentrifuge for 16 hr at 27,000 rpm and 4° C. using a Sorval TH-641rotor. Fractions (0.5 ml) were collected from the top of the sucrosegradient.

Influenza proteins in the sucrose gradient fractions were analyzed bySDS-PAGE and Western immunoblot analyses as described above in Example6. The HA and M1 proteins were found in the same sucrose gradientfractions (FIG. 6) as shown by Western blot analysis and suggested thatthe HA and M1 proteins were associated as macromolecular proteincomplexes. Also the HA and M1 proteins were found in fractionsthroughout the sucrose gradient suggesting that these recombinant viralproteins were associated with macromolecular protein complexes ofdifferent densities and compositions.

Example 10 Analysis of Recombinant Avian Influenza H9N2 VLPs andProteins by Gel Filtration Chromatography

Protein macromolecules such as VLPs and monomeric proteins migratedifferently on gel filtration or size exclusion chromatographic columnsbased on their mass size and shape. To determine whether the recombinantinfluenza proteins from sucrose gradient fractions were monomericproteins or macromolecular protein complexes such as VLPs, achromatography column (7 mm×140 mm) with a resin bed volume of 14 ml ofSepharose CL-4B (Amersham) was prepared. The size exclusion column wasequilibrated with PBS and calibrated with Dextran Blue 2000, DextranYellow, and Vitamin B12 (Amersham Pharmacia) with apparent molecularweights of 2,000,000; 20,000; and 1,357, respectively, to ascertain thecolumn void volume. Dextran Blue 2000 eluted from the column in the voidvolume (6 ml fraction) also. As expected, the recombinant influenzaprotein complexes eluted from the column in the void volume (6 mlfraction). This result was characteristic of a high molecular weightmacromolecular protein complex such as VLPs. Viral proteins in thecolumn fractions were detected by Western immunoblot analysis asdescribed above in Example 6. The M1 proteins were detected in the voidvolume fractions (FIG. 7). As expected baculovirus proteins were also inthe void volume.

Example 11 Electron Microscopy of Recombinant Influenza VLPs

To determine whether the macromolecular protein complexes isolated onsucrose gradients and containing recombinant avian influenza proteinshad morphologies similar to influenza virions, electron microscopicexamination of negatively stained samples was performed. Recombinantavian influenza A/Hong Kong/1073/99 (H9N2) protein complexes wereconcentrated and purified from culture supernatants byultracentrifugation on discontinuous sucrose gradients as described inExample 7. Aliquots of the sucrose gradient fractions were treated witha 2% glutaraldehyde in PBS, pH7.2, absorbed onto fresh dischargedplastic/carbon-coated grids, and washed with distilled water. Thesamples were stained with 2% sodium phosphotungstate, pH 6.5, andobserved using a transmission electron microscope (Philips). Electronmicrographs of negatively-stained samples of recombinant avian influenzaH9N2 protein complexes from two sucrose gradient fractions showedspherical and rod-shaped particles (FIG. 8) from two sucrose gradientfractions. The particles had different sizes (60 and 80 nm) andmorphologies. Larger complexes of both types of particles were alsodetected, as well as rod-shaped particles (FIG. 8). All observed proteincomplex structures exhibited spike like surface projections resemblinginfluenza virus HA and NA peplomers. Since the size and appearance ofthe 80 nm particles was similar to that of wild type influenza virusparticles, these structures likely represented enveloped influenza VLPs.The smaller particles of approximately 60 nm probably representedsubviral particles that differed from the above VLPs bothmorphologically and structurally.

Example 12 Analysis of Functional Characteristics of Influenza Proteinsby Hemagglutination Assay

To determine whether the purified influenza VLPs and proteins possessedfunctional activities, such as hemagglutination and neuraminidaseactivity, which were characteristic for influenza virus, the purifiedinfluenza VLPs and proteins were tested in hemagglutination andneuraminidase assays.

For the hemagglutination assay, a series of 2-fold dilutions of sucrosegradient fractions containing influenza VLPs or positive control wildtype influenza virus type A were prepared. Then they were mixed with0.6% guinea pig red blood cells in PBS (pH 7.2) and incubated at 4° C.for 1 to 16 hr. As a negative control, PBS was used. The extent ofhemagglutination was determined visually, and the highest dilution offraction capable of agglutinating guinea pig red blood cells wasdetermined (FIG. 9). The highest hemagglutination titer observed for thepurified influenza VLPs and proteins was 1:4000, which was higher thanthe titer shown by the wild type influenza control, which was 1:2000.

Example 13 Analysis of Functional Characteristics of Influenza Proteinsby Neuraminidase Assay

The amount of neuraminidase activity in influenza VLP-containing sucrosegradient fractions was determined by the neuraminidase assay. In thisassay the NA (an enzyme) acted on the substrate (fetuin) and releasedsialic acid. Arsenite reagent was added to stop enzyme activity. Theamount of sialic acid liberated was determined chemically with thethiobarbituric acid that produced a pink color in proportion to freesialic acid. The amount of color (chromophor) was measured in aspectrophotometer at wavelength 594 nm. The data, as depicted in FIG. 8,showed that a significant amount of sialic acid was produced byVLP-containing fractions of the sucrose gradients and that thesefractions corresponded to those fractions exhibiting hemagglutinationactivity.

Example 14 Immunization of BALB/c Mice with Functional HomotypicRecombinant Influenza H9N2 VLPs

The immunogenicity of the recombinant influenza VLPs was ascertained byimmunization of mice followed by Western blot analysis of immune sera.Recombinant VLPs (1 μg/injection) comprised of viral HA, NA, and M1proteins from avian influenza virus type A/Honk Kong/1073/99 andpurified on sucrose gradients were inoculated subcutaneously into thedeltoid region of ten (10) female BALB/c mice at day 0 and day 28 (FIG.11). PBS (pH 7.2) was administered similarly as a negative control intofive (5) mice. The mice were bled from the supraorbital cavity at day-1(pre-bleed), day 27 (primary bleed), and day 54 (secondary bleed). Serawere collected from blood samples following overnight clotting andcentrifugation.

For Western blot analysis, 200 ng of inactivated avian influenza virustype A H9N2 or cold-adapted avian influenza virus type A H9N2, as wellas See Blue Plus 2 pre-stained protein standards (InVitrogen), wasdenatured (95° C., 5 minutes) and subjected to electrophoresis underreduced conditions (10 mM (3-mercaptoethanol) on 4-12% polyacrylamidegradient NuPAGE gels (InVitrogen) in MES buffer at 172 volts until thebromophenol blue tracking dye disappeared. For protein gels, theelectrophoreses proteins were visualized by staining with ColloidalCoomassie Blue reagent (InVitrogen). Proteins were transferred from thegel to nitrocellulose membranes in methanol by the standard Western blotprocedure. Sera from VLP-immunized mice and rabbits immunized withinactivated avian influenza virus H9N2 (positive control sera) werediluted 1:25 and 1:100, respectively, in PBS solution (pH 7.2) and usedas primary antibody. Protein bound membranes, which were blocked with 5%casein, were reacted with primary antisera for 60 minutes at roomtemperature with constant shaking. Following washing of primary antibodymembranes with phosphate buffered saline solution containing Tween 20,secondary antisera [goat anti-murine IgG—alkaline phosphatase conjugate(1:10,000) or goat anti-rabbit IgG—alkaline phosphatase conjugate(1:10,000)] were reacted 60 minutes with the membrane. Following washingof secondary antibody membranes with phosphate buffered saline solutioncontaining Tween 20, antibody-binding proteins on the membranes werevisualized by development with the chromogenic substrate such asNBT/BCIP (InVitrogen).

The results of Western blot analysis (FIG. 12) were that proteins withmolecular weights similar to viral HA and M1 proteins (75 and 30 kd,respectively) bound to positive control sera (FIG. 12B) and sera frommice immunized with the recombinant influenza H9N2 VLPs (FIG. 12A).These results indicated that the recombinant influenza H9N2 VLPs alonewere immunogenic in mice by this route of administration.

Example 15 Kong/1073/99 (H9N2) VLP Immunogenicity And Challenge Study InBALB/c Mice

BALB/C mice were immunized with H9N2 VLPs (1 μg HA or 10 μg HA/dose),with or without 100 μg Novasome adjuvant, on day 0 and day 21 andchallenged with homologous infectious virus IN on day 57. Mice were bledon days 0, 27 and 57 with the serum assayed for anti-HA antibodies bythe hemagglutination inhibition assay (HI) using turkey RBCs, andinfluenza by ELISA. Results of this study are shown in FIG. 13 throughFIG. 16.

High titers of H9N2 antibodies were induced after a single immunization(primary) with H9N2 VLP vaccine without or with Novasomes and a dose of10 μg VLPs containing 1 μg HA (FIG. 13). Specific antibody titers wereincreased about half to one log following a booster immunization.

After immunization and a boost with 1 μg of HA in the form of H9N2 VLPsthe serum HI levels were at or above the level generally consideredprotective (log 2=5) in all animals (FIG. 14, lower left panel). H9N2VLPs formulated with Novasome adjuvant increased HI responses about 2fold following primary immunization and about 4 fold after the booster(FIG. 14, lower right panel). Purified subunit H9N2 hemagglutinin alsoinduced protective levels of HI antibodies after boosting and Novasomesagain increased HI antibody responses by about 2 fold after the primaryand 4 fold after the booster immunizations (FIG. 14, upper panels). Thelevel of HI antibody induced with 10 μg of HA given as a subunit vaccinewas equivalent to 1 μs of HA presented in the form of a VLP.

In addition, weight loss was significantly less in the mice immunizedwith H9N2 VLPs or with VLPs plus adjuvant compared to unvaccinatedcontrol animals (FIG. 15). There was no statistical difference in weightloss in the groups immunized with H9N2 VLPs and H9N2 VLPs plus Novasomeadjuvant.

Likewise, lung virus titers at 3 and 5 days post challenge with H9N2virus were significantly reduced in mice immunized with H9N2 VLPs (FIG.16). At day 3 when the influenza virus titers peak in the lung tissues,mice immunized with H9N2 VLPs plus Novasomes® had a significantlygreater reduction in virus titer compared to mice immunized with VLPsalone and the unvaccinated control mice.

Example 16 A/Fujian/411/2002 (H3N2) VLP Immunogenicity and CrossReactivity between several influenza Strains

BALB/c mice were immunized with A/Fujian/411/2002 VLPs (3.0, 0.6, 0.12and 0.24 μg HA/dose), twice IM and IN. Mice were bled on days 0 and 35.The serum was then assayed for anti-HA antibodies by thehemagglutination inhibition assay (HI) using turkey RBCs, and foranti-influenza antibodies by ELISA. Results of this study are shown onFIGS. 17A, 17B and 17C. These results indicate that an immune responsewas mounted both IM and IN against HA and NA.

Example 17 Determination of the IgG Isotypes in Mouse after Inoculationwith H3N2 VLPs

Mice were inoculated with VLPs intramuscularly and intranasal. At week 5sera was collected and assayed to distinguish between IgG isotypes.

Sera was tested on plates coated with purified HA (Protein Sciences)A/Wyoming/3/2003 using an ELISA assay. Serial five-fold dilutions ofsera was added to the wells and the plates were incubated. Next, thebiotinylated goat anti-mouse Ig, or anti-mouse IgG1, anti-mouse IgG2a,anti-mouse IgG2b and anti-mouse IgG3. Then, streptavidine-peroxidase wasadded to the wells. Bound conjugates were detected. Results areillustrated on FIGS. 18A and B. These results illustrate that IgG2a arethe most abundant isotype in an immune response against VLPs in mouse.

Example 18 A/Hong Kong/1073/99 (H9N2) VLP Dose-Ranging Study in SD Rats

SD rats (n=6 per dose) were immunized on day 0 and day 21 with purifiedA/Hong Kong/1073/99 (H9N2) VLPs diluted with PBS at neutral pH to 0.12,0.6, 3.0, and 15.0 μg HA or with PBS alone. Blood samples were takenfrom the animals on day 0, day 21, day 35 and day 49 and the serumassayed for hemagglutination inhibition assay (HI) to detect functionalantibodies able to inhibit the binding function of the HA. The dosagewas based on HA content as measured using SDS-PAGE and scanningdensitometry of purified H9N2 VLPs. Hemagglutinin inhibition assay titerresults are depicted in FIG. 19. A single 0.6 μg HA dose of H9N2 VLPs ortwo doses of 0.12 μg HA produced protectiveleVels of HI antibodies inrats. These data indicate that a lower amount of HA can induce aprotective response when said HA is part of a VLP.

Example 19 Kong/1073/99 (H9N2) VLP Immunogenicity

BALB/C mice were immunized with H9N2 VLPs (0.12, 0.6 μg HA/dose), withor without 100 μg Novasome and Alum adjuvant, on day 0 and day 21 andchallenged with homologous infectious virus IN on day 57. Mice were alsoimmunized with 3.0 and 15.0 μg HA/dose (no adjuvant). Mice were bled ondays 0, 21, 35 and 49 with the serum assayed for anti-HA antibodies bythe hemagglutination inhibition assay (HI) using turkey RBCs, andinfluenza by ELISA. Results of this study are shown in FIGS. 20 A and B.

The results indicate that a more robust overall immune response wasobserved when the VLPs were administered with an adjuvant. However, aprotective response was elicited with 0.12 μg HA/dose at week 3 whencompared to the VLPs formulation with Alum and VLPs with no adjuvant.Also in week 7, the VLPs comprising Novasomes had about 2 log increasein HI titer as compared to the VLP with Alum. The robustness of theresponse was similar to VLPs administered at 3.0 and 15.0 μg HA/dosewithout an adjuvant. These results indicate that Novasomes elicit a morerobust response as compared to Alum. In addition, a protective immuneresponse can be achieved with 25× less VLPs when said VLPs areadministered in a formulation comprising Novasomes.

Also, in the 0.6 μg HA/dose data, the Novasome formulation had an about1.5 log greater response than compared to Alum. The immune responseswere similar in magnitude to VLPs administered in 3.0 and 15.0 μgHA/dose without adjuvant. These results indicate that with an adjuvant,approximately 5× less VLPs are needed to be administered to achieve aprotective response.

Also, FIG. 20B depicts the HI titer of H9N2 VLPs using differentformulations of Novasomes. The following are the formulas used in theexperiment:

Group 1: H9N2 VLP (0.1 μg) (n=5)

Group 2: H9N2 VLP (0.1 μg) w/DCW neat) (n=5)

Group 3: H9N2 VLP (0.1 μg) w/DCW 1:3) (n=5)

Group 4: H9N2 VLP (0.1 μg) w/DCW 1:9) (n=5)

Group 5: H9N2 VLP (0.1 μg) w/DCW 1:27) (n=5)

Group 6: H9N2 VLP (0.1 μg) w/NVAX 1) (n=5)

Group 7: H9N2 VLP (0.1 μg) w/NVAX 2) (n=5)

Group 8: H9N2 VLP (0.1 μg) w/NVAX 3) (n=5)

Group 9: H9N2 VLP (0.1 μg) w/NVAX 4) (n=5)

Group 10: H9N2 VLP (0.1 μg) w/NVAX 5) (n=5)

Group 11: H9N2 VLP (0.1 μg) w/Alum-OH) (n=5)

Group 12: H9N2 VLP (0.1 μg) w/CpG) (n=5)

Group 13: PBS (0.6 μg) (n=5)

Group 14: H3 VLPs (0.6 μg) (n=5)

Group 15: H5 VLPs (0.6 μg) (n=8)

-   -   H9: (Lot#11005)    -   DCW: Novasomes (Lot#121505-2, Polyoxyethylene-2-cetyl ether,        Cholesterol, Superfined soybean oil, and Cetylpridinium        chloride)    -   NVAX 1: B35P83, MF-59 replica (Squalene, Polysorbate, and Span)    -   NVAX 2: B35P87 (Soybean Oil, Brij, Cholesterol, Pluronic F-68)    -   NVAX 3: B35P88 (Soybean Oil, Brij, Cholesterol, Pluronic F-68,        and Polyethyleneimine)    -   NVAX 4: B31P60 (Squalene, Brij, Cholesterol, Oleic acid)    -   NVAX 5: B31P63 (Soybean oil, Glyceryl monostearate, Cholesterol,        Polysorbate)    -   CpG: (Lot#1026004)    -   H5: (Lot#22406)

FIG. 21 depicts and H9N2 VLP dose response curve. This data indicatesthat a dose of VLPs at 0.6 μg HA/dose is the minimum to elicit aprotective immune response in mice after 3 weeks.

Example 20 Materials and Methods for Ferret Studies

Ferrets were purchased from Triple F Farms (FFF, Sayre, Pa.). Allferrets purchased has an HAI titer of less that 10 hemagglutinationunits. Approximately two days prior to vaccination, animals wereimplanted with a temperature transponder (BioMedic Data Systems, Inc.).Animal (6 animals/group) were vaccinated on day 0 either with (1) PBS(negative control, group one), (2) H3N2 influenza VLPs @ 15 μg of H3(group 2), (3) H3N2 influenza VLPs @ 3 μg of H3 (group 2), (4) H3N2influenza VLPs @ 0.6 μg of H3 (group 3), (5) H3N2 influenza VLPs @ 0.12μg of H3 (group 5), or (6) rH3HA @ 15 μg (group 6). On day 21 animalswere boosted with vaccine. Animals were bled on days 0 (prior tovaccination), day 21 (prior to vaccine boost), and day 42. Animals wereassessed for clinical signs of adverse vaccine effects once weeklythroughout the study period. Similar studies were performed with otherinfluenza VLPs.

HAI Levels in Ferret Sera

Ferret sera were obtained from FFF, treated with Receptor DestroyingEnzyme (RDE) and tested in a hemagglutination inhibition (HAI) assay bystandard procedures (Kendal et al. (1982)). All ferrets that were chosenfor the study tested negative (HAI=10) for pre-existing antibodies tocurrently circulating human influenza virus (A/New Calcdonia/20/99(H1N1), A panama/2007/99 (H3N2), A/Wellington/01/04 (H2N3) andB/Sichuan/379/99 and H5N1).

Ferrets

Approximately 8 month-old, influenza naïve, castrated and descented,male Fitch ferrets (Mustela putorius furo) were purchased form FFF.Animals were housed in stainless steel rabbit cages (Shor-line, KS)containing Sani-chips Laboratory Animal Bedding (P.J. Murphy ForestProducts, NJ). Ferrets were provided with Teklad Global Ferret Diet(Harlan Teklad, Wis.) and fresh water ad libitum. Pans were changedthree times each week, and cages were cleaned biweekly.

Vaccinations and Blood Collection of Ferrets

The vaccine, H3N2 influenza VLPs or H9N2 influenza VLPs and controls,for example, rH3NA (A/Wyoming/3/2003) and PBS (negative control) werestored at 4° C. prior to use. For most studies, six groups of ferrets(N-6/group) were vaccinated intramuscularly with either concentration ofvaccine or control in a volume of 0.5 ml.

Prior to blood collection and vaccination, animals were anesthetized byintramuscular injection into the inner thigh with a solution of Katamine(25 mg/kg, Atropine (0.05 mg/kg) and Xylazine (2.0 mg/kg) “KAX.” Onceunder anesthesia, ferrets were positioned in dorsal recumbency and bloodwas collected (volume between 0.5 and 1.0 ml) from the anterior venacava using a 23 gauge 1″ needle connected to a 1 cc tuberculin syringe.Blood was transferred to a tube containing a serum separator and clotactivator and allowed to clot at room temperature. Tubes werecentrifuged and sera was removed and frozen at −80° C. Blood wascollected prior to vaccination (day 0), prior to boost (day 21) and day42 and tested by HAI assay.

Monitoring of Ferrets

Temperatures were measured weekly at approximately the same timethroughout the study period. Pre-vaccination values were averaged toobtain a baseline temperature for each ferret. The change in temperature(in degrees Fahrenheit) was calculated at each time point for eachanimal. Ferrets were examined weekly for clinical signs of adversevaccine effects, including temperature, weight loss, loss of activity,nasal discharge, sneezing and diarrhea. A scoring system bases on thatdescribed by Reuman et al. (1989) was used to assess activity levelwhere 0=alert and playful; 1=alert but playful only when stimulated;2=alert by not playful when stimulated; 3=neither alert not playful whenstimulated. Based on the scores for each animal in a group, a relativeinactivity index was calculated as Σ(day0−Day 42)[activityscore+1]/Σ(day0−Day 42), where n equals the total number ofobservations. A value of 1 was added to each base score so that a scoreof “0” could be divided by a denominator, resulting in an index value of1.0.

Serum Preparations

Sera generally have low levels of non-specific inhibitors onhemagglutination. To inactivate these non-specific inhibitors, sera weretreated with (RDE) prior to being tested. Briefly, three part RDE wasadded to one part sera and incubated overnight at 37° C. RDE wasinactivated by incubation at 56° C. for approximately 30 minutes.Following inactivation of RDE, PBS was added to the sample for a finalserum dilution of 1:10 (RDE-Tx). The diluted RDE-Tx sera was stored at4° C. prior to testing (for 7 days) or stored at −20° C.

Preparation Turkey Erythrocytes:

Human influenza viruses bind to sialic acid receptors containingN-acetylneuraminic acid α 2,6-galactose linkages. Avian influenzaviruses bind to sialic acid receptors containing N-acetylneuraminic acidα 2,3 galactose (α 2,3 linkages) and express both α 2,3 and α 2,6linkages. Turkey erythocytes (TRBC) are used for the HAI assay sinceA/Fujian is a human influenza virus. The TRBCs adjusted with PBS toachieve a 0.5% vol/vol suspension. The cells are kept at 4° C. and usedwithin 72 hours of preparation.

HAI Assay

The HAI assay was adapted from the CDC laboratory-based influenzasurveillance manual (Kendal et al. (1982) Concepts and procedures forlaboratory based influenza surveillance, U.S. Department of Health andHuman Services, Public Health Service, Centers for Disease Control,Atlanta, Ga., herein incorporated by reference in its entirety for allpurposes). RDE-Tx sera was serially two-fold diluted in v-bottommicrotiter plates. An equal volume of virus adjusted, adjusted toapproximately 8 HAU/50 ul was added to each well. The plates werecovered and incubated at room temperature for 15 minutes followed by theaddition of 0.5% TRBC. The plates were mixed by agitation, covered, andthe TRBC were allowed to settle for 30 minutes at room temperature. TheHAI titer was determined by the reciprocal dilution of the last rowwhich contained non-agglutinated TRBC. Positive and negative serumcontrols were included for each plate.

Example 21 A/Hong Kong/1073/99 (H9N2) VLP Dose-Ranging Study in Ferrets

Ferrets, serologically negative by hemagglutination inhibition forinfluenza viruses, were used to assess the antibody and HI titer afteran inoculation with H9N2 VLPs. Ferrets were bled on days 0, and 21 dayswith the serum assayed for anti-HA antibodies by the hemagglutinationinhibition assay (HI) using turkey RBCs, and for anti-influenzaantibodies by ELISA. Results are illustrated in FIG. 22. These resultsshow HI titers corresponding to protective antibody levels at VLP dosesof 1.5 and 15 μg.

Example 21 Vaccination of H3N2 VLPs in Ferrets

Ferrets were vaccinated at day 0, and given a boost on day 21 withdifferent strains of H3N2 VLPs at different dosages (HA dosages of 0.12,0.6, 3.0, 15.0 μg). The positive control was rH3HA at 15 μg and PBSalone is the negative control. Sera, as described above, were taken fromthe ferrets on day 0 prior to vaccination, day 21 (prior to boost) andday 42. An HI assay was conducted on the serum samples to determine ifthere was an immune response against the VLPs. These data areillustration on FIG. 23. These data indicate that H3N2 VLPs, whenintroduced into ferrets, do induce an immune response. Thus, the H3N2VLPs are immunogenic in ferrets.

Example 22 RT-PCR and Cloning of HA, NA, and M1 Genes of InfluenzaA/Indonesia/5/05 (H5N1) Virus

Clade 2 influenza virus, strain A/Indonesia/5/05 (H5N1) viral RNA wasextracted using Trizol LS (Invitrogen, Carlsbad, Calif.) under BSL-3containment conditions. Reverse transcription (RT) and PCR wereperformed on extracted viral RNA using the One-Step RT-PCR system(Invitrogen) with gene-specific oligonucleotide primers. The followingprimer pairs were used for the synthesis of the H5N1 hemagglutinin (HA),neuraminidase (NA), and matrix (M1) genes, respectively:

(SEQ ID. 4) 5′-AACGGTCCGATGGAGAAAATAGTGCTTCTTC-3′ and (SEQ ID. 5)5′-AAAGCTTTTAAATGCAAATTCTGCATTGTAACG-3′ (HA); (SEQ ID. 6)5′-AACGGTCCGATGAATCCAAATCAGAAGATAAT-3′ and (SEQ ID. 7)5′-AAAGCTTCTACTTGTCAATGGTGAATGGCAAC-3′ (NA); and (SEQ ID. 8)5′-AACGGTCCGATGAGTCTTCTAACCGAGGTC-3′ and (SEQ ID. 9)5′-AAAGCTTTCACTTGAATCGCTGCATCTGCAC-3′ (M1) (ATG codons are underlined).

Following RT-PCR, cDNA fragments containing influenza HA, NA, and M1genes with molecular weights of 1.7, 1.4, and 0.7 kB, respectively, werecloned into the pCR2.1-TOPO vector (Invitrogen). The nucleotidesequences of the HA, NA, and M1 genes were determined by DNA sequencing.A similar strategy was followed for cloning a clade 1 H5N1 influenzavirus from Vietnam/1203/2003.

Example 23 Generation of Recombinant Baculoviruses Comprising H5N1

The HA gene was cloned as a RsrII-HindIII DNA fragment (1.7 kb)downstream of the AcMNPV polyhedrin promoter within pFastBac1 bacmidtransfer vector (Invitrogen) digested with RsrII and HindIIII.Similarly, the NA and M1 genes were cloned as EcoRI-HindIII DNAfragments (1.4 and 0.8 kb, respectively) into EcoRI-HindIII-digestedpFastBac1 plasmid DNA. The three resulting baculovirus transfer plasmidspHA, pNA, and pM1 containing influenza A/Indonesia/5/05 (H5N1) virus HA,NA, and M1 genes, respectively, were used to generate recombinantbacmids.

Bacmids were produced by site-specific homologous recombinationfollowing transformation of bacmid transfer plasmids containinginfluenza genes into E. coli DH10Bac competent cells, which containedthe AcMNPV baculovirus genome (Invitrogen). The recombinant bacmid DNAwas transfected into the Sf9 insect cells.

Nucleotide sequences of the Indonesia/5/05 HA, NA, and M1 genes.

HA (SEQ ID. 10)ATGGAGAAAATAGTGCTTCTTCTTGCAATAGTCAGTCTTGTTAAAAGTGATCAGATTTGCATTGGTTACCATGCAAACAATTCAACAGAGCAGGTTGACACAATCATGGAAAAGAACGTTACTGTTACACATGCCCAAGACATACTGGAAAAGACACACAACGGGAAGCTCTGCGATCTAGATGGAGTGAAGCCTCTAATTTTAAGAGATTGTAGTGTAGCTGGATGGCTCCTCGGGAACCCAATGTGTGACGAATTCATCAATGTACCGGAATGGTCTTACATAGTGGAGAAGGCCAATCCAACCAATGACCTCTGTTACCCAGGGAGTTTCAACGACTATGAAGAACTGAAACACCTATTGAGCAGAATAAACCATTTTGAGAAAATTCAAATCATCCCCAAAAGTTCTTGGTCCGATCATGAAGCCTCATCAGGAGTGAGCTCAGCATGTCCATACCTGGGAAGTCCCTCCTTTTTTAGAAATGTGGTATGGCTTATCAAAAAGAACAGTACATACCCAACAATAAAGAAAAGCTACAATAATACCAACCAAGAAGATCTTTTGGTACTGTGGGGAATTCACCATCCTAATGATGCGGCAGAGCAGACAAGGCTATATCAAAACCCAACCACCTATATTTCCATTGGGACATCAACACTAAACCAGAGATTGGTACCAAAAATAGCTACTAGATCCAAAGTAAACGGGCAAAGTGGAAGGATGGAGTTCTTCTGGACAATTTTAAAACCTAATGATGCAATCAACTTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGCATACAAAATTGTCAAGAAAGGGGACTCAGCAATTATGAAAAGTGAATTGGAATATGGTAACTGCAACACCAAGTGTCAAACTCCAATGGGGGCGATAAACTCTAGTATGCCATTCCACAACATACACCCTCTCACCATCGGGGAATGCCCCAAATATGTGAAATCAAACAGATTAGTCCTTGCAACAGGGCTCAGAAATAGCCCTCAAAGAGAGAGCAGAAGAAAAAAGAGAGGACTATTTGGAGCTATAGCAGGTTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCATAGCAATGAGCAGGGGAGTGGGTACGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAGTCACCAATAAGGTCAACTCAATCATTGACAAAATGAACACTCAGTTTGAGGCCGTTGGAAGGGAATTTAATAACTTAGAAAGGAGAATAGAGAATTTAAACAAGAAGATGGAAGACGGGTTTCTAGATGTCTGGACTTATAATGCCGAACTTCTGGTTCTCATGGAAAATGAGAGAACTCTAGACTTTCATGACTCAAATGTTAAGAACCTCTACGACAAGGTCCGACTACAGCTTAGGGATAATGCAAAGGAGCTGGGTAACGGTTGTTTCGAGTTCTATCACAAATGTGATAATGAATGTATGGAAAGTATAAGAAACGGAACGTACAACTATCCGCAGTATTCAGAAGAAGCAAGATTAAAAAGAGAGGAAATAAGTGGGGTAAAATTGGAATCAATAGGAACTTACCAAATACTGTCAATTTATTCAACAGTGGCGAGTTCCCTAGCACTGGCAATCATGATGGCTGGTCTATCTTTATGGATGTGCTCCAATGGATCGTTACAATGCAGAATTTGCATTTAA NA (SEQ ID. 11)ATGAATCCAAATCAGAAGATAATAACCATTGGATCAATCTGTATGGTAATTGGAATAGTTAGCTTAATGTTACAAATTGGGAACATGATCTCAATATGGGTCAGTCATTCAATTCAGACAGGGAATCAACACCAAGCTGAATCAATCAGCAATACTAACCCTCTTACTGAGAAAGCTGTGGCTTCAGTAACATTAGCGGGCAATTCATCTCTTTGCCCCATTAGAGGATGGGCTGTACACAGTAAGGACAACAATATAAGGATCGGTTCCAAGGGGGATGTGTTTGTTATTAGAGAGCCGTTCATCTCATGCTCCCACCTGGAATGCAGAACTTTCTTCTTGACTCAGGGAGCCTTGCTGAATGACAAGCACTCCAACGGGACTGTCAAAGACAGAAGCCCTCACAGAACATTAATGAGTTGTCCTGTGGGTGAGGCTCCCTCTCCATATAACTCAAGGTTTGAGTCTGTTGCTTGGTCAGCAAGTGCTTGCCATGATGGCACCAGTTGGTTGACAATTGGAATTTCTGGCCCAGACAATGAGGCTGTGGCTGTATTGAAATACAATGGCATAATAACAGACACTATCAAGAGTTGGAGGAACAACATACTGAGAACTCAAGAGTCTGAATGTGCATGTGTAAATGGCTCTTGCTTTACTGTAATGACTGATGGACCAAGTGATGGGCAGGCATCATATAAGATCTTCAAAATGGAAAAAGGAAAAGTGGTCAAATCAGTCGAATTGGATGCTCCTAATTATCACTATGAGGAATGCTCCTGTTATCCTGATGCCGGCGAAATCACATGTGTTTGCAGGGATAATTGGCATGGCTCAAATAGGCCATGGGTATCTTTCAATCAAAATTTGGAGTATCAAATAGGATATATATGCAGTGGAGTTTTCGGAGACAATCCACGCCCCAATGATGGAACAGGTAGTTGTGGCCCGATGTCCCCTAACGGGGCATATGGGGTAAAAGGGTTTTCATTTAAATACGGCAATGGTGTTTGGATCGGGAGAACCAAAAGCACTAATTCCAGGAGCGGCTTTGAAATGATTTGGGATCCAAATGGGTGGACTGGAACGGACAGTAGCTTTTCAGTGAAACAAGATATAGTAGCAATAACTGATTGGTCAGGATATAGCGGGAGTTTTGTCCAGCATCCAGAACTGACAGGATTAGATTGCATAAGACCTTGTTTCTGGGTTGAGTTAATCAGAGGGCGGCCCAAAGAGAGCACAATTTGGACTAGTGGGAGCAGCATATCTTTTTGTGGTGTAAATAGTGACACTGTGAGTTGGTCTTGGCCAGACGGTGCTGAGTTGCCATTCACCATTGACAAGTAG M1 (SEQ ID. 12)ATGAGTCTTCTAACCGAGGTCGAAACGTACGTTCTCTCTATCATCCCGTCAGGCCCCCTCAAAGCCGAGATCGCGCAGAAACTTGAAGATGTCTTTGCAGGAAAGAACACCGATCTCGAGGCTCTCATGGAGTGGCTGAAGACAAGACCAATCCTGTCACCTCTGACTAAAGGGATTTTGGGATTTGTATTCACGCTCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGCTTTGTCCAGAATGCCCTAAATGGAAATGGAGATCCAAATAATATGGATAGGGCAGTTAAGCTATATAAGAAGCTGAAAAGAGAAATAACATTCCATGGGGCTAAAGAGGTTTCACTCAGCTACTCAACCGGTGCACTTGCCAGTTGCATGGGTCTCATATACAACAGGATGGGAACGGTGACTACGGAAGTGGCTTTTGGCCTAGTGTGTGCCACTTGTGAGCAGATTGCAGATTCACAGCATCGGTCTCACAGGCAGATGGCAACTATCACCAACCCACTAATCAGGCATGAAAACAGAATGGTGCTGGCCAGCACTACAGCTAAGGCTATGGAGCAGATGGCGGGATCAAGTGAGCAGGCAGCGGAAGCCATGGAGGTCGCTAATCAGGCTAGGCAGATGGTGCAGGCAATGAGGACAATTGGAACTCATCCTAACTCTAGTGCTGGTCTGAGAGATAATCTTCTTGAAAATTTGCAGGCCTACCAGAAACGAATGGGAGTGCAGATGCAGCGATTCAAGTGA

One cloned HA gene, pHA5, contained two nucleotide changes, nt #1172 andnt #1508 (in the wt), as compared to the wild-type HA gene sequence. Asimilar strategy was followed for constructing and creating clade 1 H5N1influenza virus from Vietnam/1203/2003 VLPs (see below). The alignmentsof pHA5 nucleotide and amino acid sequences follow.

Amino Acid Sequence Alignment of Hemagglutinin

Example 26 Generation of Influenza A/Indonesia/5/05 HA, NA, and M1 GenesOptimized for Efficient Expression in Sf9 Cells

The following polypeptides were derived from codon-optimized nucleotidescorresponding to the Indonesia/5/05 HA gene (see example 31). Thecodon-optimized nucleotides were designed and produced (Geneart GMBH,Regensburg, FRG) according to the methods disclosed in US patentpublication 2005/0118191, herein incorporated by reference in itsentirety for all proposes. See Example 31 for nucleic acid sequences

Vac2-hac-opt (unmodified aa sequence) (SEQ ID 27)MEKIVLLLAI VSLVKSDQIC IGYHANNSTE QVDTIMEKNVTVTHAQDILE KTHNGKLCDL DGVKPLILRD CSVAGWLLGNPMCDEFINVP EWSYIVEKAN PTNDLCYPGS FNDYEELKHLLSRINHFEKI QIIPKSSWSD HEASSGVSSA CPYLGSPSFFRNVVWLIKKN STYPTIKKSY NNTNQEDLLV LWGIHHPNDAAEQTRLYQNP TTYISIGTST LNQRLVPKIA TRSKVNGQSGRMEFFWTILK PNDAINFESN GNFIAPEYAY KIVKKGDSAIMKSELEYGNC NTKCQTPMGA INSSMPFHNI HPLTIGECPKYVKSNRLVLA TGLRNSPQRE SRRKKRGLFG AIAGFIEGGWQGMVDGWYGY HHSNEQGSGY AADKESTQKA IDGVTNKVNSIIDKMNTQFE AVGREFNNLE RRIENLNKKM EDGFLDVWTYNAELLVLMEN ERTLDFHDSN VKNLYDKVRL QLRDNAKELGNGCFEFYHKC DNECMESIRN GTYNYPQYSE EARLKREEISGVKLESIGTY QILSIYSTVA SSLALAIMMA GLSLWMCSNG SLQCRICI* Vac2-hac-spc-opt(modified, signal peptide from Chitinase, underlined) (SEQ ID 28)Mplykllnvlwlvavsnaip DQICIGYHANNSTE QVDTIMEKNVTVTHAQDILE KTHNGKLCDL DGVKPLILRD CSVAGWLLGNPMCDEFINVP EWSYIVEKAN PTNDLCYPGS FNDYEELKHLLSRINHFEKI QIIPKSSWSD HEASSGVSSA CPYLGSPSFFRNVVWLIKKN STYPTIKKSY NNTNQEDLLV LWGIHHPNDAAEQTRLYQNP TTYISIGTST LNQRLVPKIA TRSKVNGQSGRMEFFWTILK PNDAINFESN GNFIAPEYAY KIVKKGDSAIMKSELEYGNC NTKCQTPMGA INSSMPFHNI HPLTIGECPKYVKSNRLVLA TGLRNSPQRE SRRKKRGLFG AIAGFIEGGWQGMVDGWYGY HHSNEQGSGY AADKESTQKA IDGVTNKVNSIIDKMNTQFE AVGREFNNLE RRIENLNKKM EDGFLDVWTYNAELLVLMEN ERTLDFHDSN VKNLYDKVRL QLRDNAKELGNGCFEFYHKC DNECMESIRN GTYNYPQYSE EARLKREEISGVKLESIGTY QILSIYSTVA SSLALAIMMA GLSLWMCSNG SLQCRICI* Vac2-hac-sph9-opt(modified, signal peptide from H9, underlined) (SEQ ID 29)METISLITIL LVVTASNA DQICIGYHANNSTE QVDTIMEKNVTVTHAQDILE KTHNGKLCDL DGVKPLILRD CSVAGWLLGNPMCDEFINVP EWSYIVEKAN PTNDLCYPGS FNDYEELKHLLSRINHFEKI QIIPKSSWSD HEASSGVSSA CPYLGSPSFFRNVVWLIKKN STYPTIKKSY NNTNQEDLLV LWGIHHPNDAAEQTRLYQNP TTYISIGTST LNQRLVPKIA TRSKVNGQSGRMEFFWTILK PNDAINFESN GNFIAPEYAY KIVKKGDSAIMKSELEYGNC NTKCQTPMGA INSSMPFHNI HPLTIGECPKYVKSNRLVLA TGLRNSPQRE SRRKKRGLFG AIAGFIEGGWQGMVDGWYGY HHSNEQGSGY AADKESTQKA IDGVTNKVNSIIDKMNTQFE AVGREFNNLE RRIENLNKKM EDGFLDVWTYNAELLVLMEN ERTLDFHDSN VKNLYDKVRL QLRDNAKELGNGCFEFYHKC DNECMESIRN GTYNYPQYSE EARLKREEISGVKLESIGTY QILSIYSTVA SSLALAIMMA GLSLWMCSNG SLQCRICI* Vac2-hac-cs-opt(- is the modified cleavage site) (SEQ ID 30)MEKIVLLLAI VSLVKSDQIC IGYHANNSTE QVDTIMEKNVTVTHAQDILE KTHNGKLCDL DGVKPLILRD CSVAGWLLGNPMCDEFINVP EWSYIVEKAN PTNDLCYPGS FNDYEELKHLLSRINHFEKI QIIPKSSWSD HEASSGVSSA CPYLGSPSFFRNVVWLIKKN STYPTIKKSY NNTNQEDLLV LWGIHHPNDAAEQTRLYQNP TTYISIGTST LNQRLVPKIA TRSKVNGQSGRMEFFWTILK PNDAINFESN GNFIAPEYAY KIVKKGDSAIMKSELEYGNC NTKCQTPMGA INSSMPFHNI HPLTIGECPKYVKSNRLVLA TGLRNSPQRE S----RGLFG AIAGFIEGGWQGMVDGWYGY HHSNEQGSGY AADKESTQKA IDGVTNKVNSIIDKMNTQFE AVGREFNNLE RRIENLNKKM EDGFLDVWTYNAELLVLMEN ERTLDFHDSN VKNLYDKVRL QLRDNAKELGNGCFEFYHKC DNECMESIRN GTYNYPQYSE EARLKREEISGVKLESIGTY QILSIYSTVA SSLALAIMMA GLSLWMCSNG SLQCRICI*

The following polypeptides corresponding to unmodified, codon-optimizedNA and M1 genes where also synthesized.

Vac2-naj-opt (neuraminidase) (SEQ ID 31)MNPNQKIITI GSICMVIGIV SLMLQIGNMI SIWVSHSIQTGNQHQAESIS NTNPLTEKAV ASVTLAGNSS LCPIRGWAVHSKDNNIRIGS KGDVFVIREP FISCSHLECR TFFLTQGALLNDKHSNGTVK DRSPHRTLMS CPVGEAPSPY NSRFESVAWSASACHDGTSW LTIGISGPDN EAVAVLKYNG IITDTIKSWRNNILRTQESE CACVNGSCFT VMTDGPSDGQ ASYKIFKMEKGKVVKSVELD APNYHYEECS CYPDAGEITC VCRDNWHGSNRPWVSFNQNL EYQIGYICSG VFGDNPRPND GTGSCGPMSPNGAYGVKGFS FKYGNGVWIG RTKSTNSRSG FEMIWDPNGWTGTDSSFSVK QDIVAITDWS GYSGSFVQHP ELTGLDCIRPCFWVELIRGR PKESTIWTSG SSISFCGVNS DTVSWSWPDG AELPFTIDK*Vac2-mc-opt(matrix) (SEQ ID 32)MSLLTEVETY VLSIIPSGPL KAEIAQKLED VFAGKNTDLEALMEWLKTRP ILSPLTKGIL GFVFTLTVPS ERGLQRRRFVQNALNGNGDP NNMDRAVKLY KKLKREITFH GAKEVSLSYSTGALASCMGL IYNRMGTVTT EVAFGLVCAT CEQIADSQHRSHRQMATITN PLIRHENRMV LASTTAKAME QMAGSSEQAAEAMEVANQAR QMVQAMRTIG THPNSSAGLR DNLLENLQAY QKRMGVQMQR FK*

The synthetic, codon-optimized HA, NA, and M1 genes were subcloned intopFastBac1 transfer plasmid using BamHI and HindIII sites, as describedabove. Recombinant bacmids for expression in Sf9 cells of synthetic HA,NA, M1 genes were generated as described above, using E. coli strainDH10Bac (Invitrogen).

Example 24 Cloning of Clade 1 A/Viet Nam/1203/04 (H5N1) Influenza Virusby RT-PCR

The HA, NA and M1 genes were cloned by RT-PCR according to the abovedescribes method. The below sequences are comparisons of the publishedgene compared to the cloned genes.

The HA gene for Clade 1 A/Viet Nam/1203/04 (H5N1)

Comparison of the NA Genes.

Comparisons of the M1 Genes.

All the sequences were cloned and analyzed according to the disclosedmethods above.

Example 25 Generation of Clade 1 H5N1 Influenza A/Viet Nam/1203/04 HA,NA, and M1 Genes Optimized for Efficient Expression in Sf9 Cells

The following polypeptides were derived from codon-optimized nucleotidescorresponding to A/Viet Nam/1203/04. The nucleotides were designed andsynthesized (Geneart GMBH, Regensburg, FRG) as disclosed above (seeExample 24).

VN1203-ha-cs-opt  (modified cleavage site, underlined)  (SEQ ID 33)MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKNSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRET----RGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRI CI*VN1203-ha-spc-opt   (modified signal peptide, underlined) (SEQ ID 34)Mplykllnvlwlvavsnaip DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKNSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRI CI*VN1203-ha-sph9-opt (The signal peptide and cleavage site are shaded)(SEQ ID 35)

AQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKNSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLR

GVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRI CI*

Example 26 H5N1 Vietnam/1203/2003 VLP Immunogenicity (Extreme DoseSparing)

BALB/C mice were immunized intramuscularly and intranasally with H5N1VLPs at very low doses of VLPs (0.2, 0.04, 0.008, 0.0016 μg HA/dose),Mice were bled on days 0, 21 and 35. The mice were given a boost on day21. The serum was assayed for anti-HA antibodies by the hemagglutinationinhibition assay (HI) using turkey RBCs and influenza virus using anELISA. Results of this study are shown in FIGS. 24 and 25.

The results indicate that a robust overall immune response was observedwhen the VLPs were administered intramuscularly at very lose doses. Therobustness of the response was similar to control at 3.0 and 0.6 μgHA/dose. These data show see a true dose response and the antibodyresponse to 0.2 μg of VLP is greater than 3.0 μg of rHA protein.Although the response was not as robust for the intranasaladministration, a dose of VLPs at 0.2 μg HA/dose did induce a robustresponse. The ELISA titer with the 0.2 μg dose in this experiment issimilar to the 0.12 μg dose of the H3N2 VLP vaccine in previousexperiments, see above.

Example 27 Challenge Studies

After inoculating BALB/c mice with VLPs at concentrations of 3 μg, 0.6μg 0.12 μg and 0.02 μg of H3N2 VLPs intramuscularly and intranasally(total HA dose), mice were challenged with influenza virusA/Aichi/268×31. The results of this study are shown on FIGS. 27 and 28.These data show that there is a decrease in weight in all vaccinatedanimals, however the animals that were vaccinated with 3.0 μg and 0.12μg of VLPs recovered quicker than the other animals in bothintramuscular and intranasal vaccinations. The intranasal doses providedenhanced protection.

Example 29 Challenge Studies (Ferrets)

In this study, ferrets were vaccinated with H9N2 VLPs. There were atotal of 18 ferrets in the challenge study: 6 mock vaccinated, 6vaccinated with medium dose (1.5 μg), and 6 vaccinated with high dose(15.0 μg) intramuscularly. Next, ferrets were challenged with 10⁶ EID₅₀of A/HK/1073/99 intranasally. Nasal washes were collected on days 1, 3,5 and 7. The virus in the nasal washes was titered on days 3, 5 and 7for all animals. These data are represented on Table 2 and in FIG. 29.These data show that by day 7, all of the vaccinated animals had nodetectable virus in nasal washes while the mock group had detectableviral titers.

TABLE 2 Wild Type Virus Titers (log 10/ml) in Ferrets after viralchallenge Ferret Day 3 Day 5 Day 7 Group: Placebo Mock Control (n = 6)4512 7 5.5 3.5 4524 6.5 6.75 1.98 4525 7.5 6.5 6.75 4526 7.5 7.25 3.54527 6.75 7.25 2.5 4528 7.5 6.25 2.75 Mean 7.125 6.583333 3.496667 Std.Dev. 0.44017 0.66458 1.699137 Group: Low Dose 3916 6.75 2.75 1.5 39177.5 5.5 1.5 3918 7.5 6.5 1.5 3919 5.5 3 1.5 3920 6.75 2.25 1.5 3921 6.53.5 1.5 Avg 6.75 3.916667 1.5 Std Dev 0.74162 1.693123 0 Group: HighDose 3922 6.5 2.75 1.5 3923 6.25 3.75 1.5 3924 5.75 1.5 1.5 3925 6.54.75 1.5 3926 6.25 3.5 1.5 3927 5.75 1.5 1.5 Avg. 6.166667 2.958333 1.5Std Dev 0.341565 1.298236 0

Example 30 Mice Intramuscular and Intranasal Inoculation Studies

Mice were inoculated with A/Fujian/411/2002 (H3N2) VLPs atconcentrations of 3 μg, 0.6 μg 0.12 μg or 0.024 μg (total HA dose)intramuscularly or intranasally at day 0 and were boosted 3 weeks later.Control mice were inoculated with formalin inactivated A/Wyoming(Fujian-Like, vaccine strain) or PBS. Sera were collected from theinoculated mice at weeks 0, 3, 5 and 8. The collected sera were assayedfor anti-HA antibodies by the hemagglutination inhibition assay (HI) foranti-influenza antibodies by ELISA. The assay was conducted usingA/Fujian/411/2002, A/Panama/2007/99, A/Wyoming/3/03 and A/NewYork/55/2004 influenza virus strains of H3N2. Results of this study areshown on FIGS. 30 A-H. These data indicate the H3N2 VLPs inducedantibodies against the parent A/Fujian/411/2002 strains of influenzavirus and against other H3N2 strains. These data also indicate that thetiters in intranasally inoculated mice rise later than intramuscularlyinoculated mice. However, the intranasal titers are higher thanintramuscular titers after about 8 weeks. In addition, titers to theinactivated virus antigen appear to be comparable to the VLP atequivalent doses following intramuscular inoculation. However, theinactivated antigen does not appear to be as immunogenic followingintranasal inoculation, nor is it as broadly protective followingintranasal inoculation.

Example 31 Generation of Clade 2 H5N1 Influenza HA, NA, and M1 GenesOptimized for Efficient Expression in Sf9 Cells

The following optimized nucleotides and polypeptides corresponding toHA, NA and M1 of Clade 2 H5N1 viruses, A A/Indonesia/5/05, A/Bar headedgoose/Qinghai/1A/2005 and A/Anhui/1/2005, were designed and synthesized(Geneart GMBH, Regensburg, FRG) as disclosed above. The optimizednucleotides and polypeptides are listed below. In order to make VLPs,A/Anhui HA can be expressed with A/Indonesia NA and M1. For VLPscomprising A/Quinghai HA and NA, A/Indonesia M1 gene can be co-expressedwith A/Quinghai HA and NA.

A/INDONESIA/5/05A/INDONESIA Optimized HA (Start and stop codon are underlined) (SEQ ID 42) GGTACCGGATCCGCCACCATGGAGAAGATCGTGCTGCTGCTGGCTATCGTGTCCCTGGTGAAGTCCGACCAGATCTGCATCGGTTACCACGCTAACAACTCCACCGAGCAGGTGGACACCATCATGGAGAAGAACGTCACCGTGACCCACGCTCAGGACATCCTCGAAAAGACCCACAACGGCAAGCTGTGCGACCTGGACGGTGTCAAGCCCCTGATCCTGCGTGACTGCTCCGTGGCTGGTTGGCTGCTGGGTAACCCCATGTGCGACGAGTTCATCAACGTGCCCGAGTGGTCCTACATCGTGGAGAAGGCTAACCCCACCAACGACCTGTGCTACCCCGGTTCCTTCAACGACTACGAGGAGCTGAAGCACCTGCTGTCCCGTATCAACCACTTCGAGAAGATCCAGATCATCCCCAAGTCCTCTTGGTCCGACCACGAGGCTTCCTCCGGTGTCTCCTCCGCTTGCCCCTACCTGGGTTCCCCCTCCTTCTTCCGTAACGTGGTGTGGCTGATCAAGAAGAACTCCACCTACCCCACCATCAAGAAGTCCTACAACAACACCAACCAGGAGGACCTGCTGGTCCTGTGGGGTATCCACCACCCCAACGACGCTGCCGAGCAGACCCGTCTGTACCAGAACCCCACCACCTACATCTCCATCGGCACCTCCACCCTGAACCAGCGTCTGGTGCCCAAGATCGCTACCCGTTCCAAGGTGAACGGCCAGTCCGGTCGTATGGAGTTCTTCTGGACCATCCTGAAGCCTAACGACGCTATCAACTTCGAGTCCAACGGCAACTTCATCGCTCCCGAGTACGCTTACAAGATCGTGAAGAAGGGCGACTCCGCTATCATGAAGTCCGAGCTGGAGTACGGTAACTGCAACACCAAGTGCCAGACCCCCATGGGTGCTATCAACTCCTCCATGCCCTTCCACAACATCCACCCCCTGACCATCGGCGAGTGCCCCAAGTACGTGAAGTCCAACCGTCTGGTGCTGGCTACCGGTCTGCGTAACTCCCCCCAGCGCGAGTCCCGTCGTAAGAAGCGTGGTCTGTTCGGCGCTATCGCTGGTTTCATCGAGGGCGGTTGGCAGGGCATGGTGGACGGATGGTACGGTTACCACCACTCTAACGAGCAGGGTTCCGGTTACGCTGCTGACAAGGAGTCCACCCAGAAGGCTATCGACGGCGTCACCAACAAGGTGAACTCCATCATCGACAAGATGAACACCCAGTTCGAGGCTGTGGGTCGTGAGTTCAACAACCTCGAGCGTCGTATCGAGAACCTGAACAAGAAGATGGAGGACGGTTTCCTGGACGTGTGGACCTACAACGCCGAGCTGCTGGTGCTGATGGAGAACGAGCGTACCCTGGACTTCCACGACTCCAACGTGAAGAACCTGTACGACAAGGTCCGCCTGCAGCTGCGTGACAACGCTAAGGAGCTGGGTAACGGTTGCTTCGAGTTCTACCACAAGTGCGACAACGAGTGCATGGAGTCCATCCGTAACGGCACCTACAACTACCCCCAGTACTCCGAGGAGGCTCGTCTGAAGCGTGAGGAGATCTCCGGCGTGAAGCTCGAGTCCATCGGAACCTACCAGATCCTGTCCATCTACTCCACCGTGGCTTCCTCCCTGGCTCTGGCTATCATGATGGCTGGTCTGTCCCTG

A/INDONESIA HA Protein Sequence  (SEQ ID 43)MEKIVLLLAI VSLVKSDQIC IGYHANNSTE QVDTIMEKNV TVTHAQDILEKTHNGKLCDL DGVKPLILRD CSVAGWLLGN PMCDEFINVP EWSYIVEKANPTNDLCYPGS FNDYEELKHL LSRINHFEKI QIIPKSSWSD HEASSGVSSACPYLGSPSFF RNVVWLIKKN STYPTIKKSY NNTNQEDLLV LWGIHHPNDAAEQTRLYQNP TTYISIGTST LNQRLVPKIA TRSKVNGQSG RMEFFWTILKPNDAINFESN GNFIAPEYAY KIVKKGDSAI MKSELEYGNC NTKCQTPMGAINSSMPFHNI HPLTIGECPK YVKSNRLVLA TGLRNSPQRE SRRKKRGLFGAIAGFIEGGW QGMVDGWYGY HHSNEQGSGY AADKESTQKA IDGVTNKVNSIIDKMNTQFE AVGREFNNLE RRIENLNKKM EDGFLDVWTY NAELLVLMENERTLDFHDSN VKNLYDKVRL QLRDNAKELG NGCFEFYHKC DNECMESIRNGTYNYPQYSE EARLKREEIS GVKLESIGTY QILSIYSTVA SSLALAIMMAGLSLWMCSNG SLQCRICI A/INDONESIA Optimized HA (cleavage site deleted)(Start and stop codon are underlined) (SEQ ID 44)GGATCCGCCACCATGGAGAAGATCGTGCTGCTGCTGGCTATCGTGTCCCTGGTGAAGTCCGACCAGATCTGCATCGGTTACCACGCTAACAACTCCACCGAGCAGGTGGACACCATCATGGAGAAGAACGTCACCGTGACCCACGCTCAGGACATCCTCGAAAAGACCCACAACGGCAAGCTGTGCGACCTGGACGGTGTCAAGCCCCTGATCCTGCGTGACTGCTCCGTGGCTGGTTGGCTGCTGGGTAACCCCATGTGCGACGAGTTCATCAACGTGCCCGAGTGGTCCTACATCGTGGAGAAGGCTAACCCCACCAACGACCTGTGCTACCCCGGTTCCTTCAACGACTACGAGGAGCTGAAGCACCTGCTGTCCCGTATCAACCACTTCGAGAAGATCCAGATCATCCCCAAGTCCTCTTGGTCCGACCACGAGGCTTCCTCCGGTGTCTCCTCCGCTTGCCCCTACCTGGGTTCCCCCTCCTTCTTCCGTAACGTGGTGTGGCTGATCAAGAAGAACTCCACCTACCCCACCATCAAGAAGTCCTACAACAACACCAACCAGGAGGACCTGCTGGTCCTGTGGGGTATCCACCACCCCAACGACGCTGCCGAGCAGACCCGTCTGTACCAGAACCCCACCACCTACATCTCCATCGGCACCTCCACCCTGAACCAGCGTCTGGTGCCCAAGATCGCTACCCGTTCCAAGGTGAACGGCCAGTCCGGTCGTATGGAGTTCTTCTGGACCATCCTGAAGCCTAACGACGCTATCAACTTCGAGTCCAACGGCAACTTCATCGCTCCCGAGTACGCTTACAAGATCGTGAAGAAGGGCGACTCCGCTATCATGAAGTCCGAGCTGGAGTACGGTAACTGCAACACCAAGTGCCAGACCCCCATGGGTGCTATCAACTCCTCCATGCCCTTCCACAACATCCACCCCCTGACCATCGGCGAGTGCCCCAAGTACGTGAAGTCCAACCGTCTGGTGCTGGCTACCGGTCTGCGTAACTCCCCCCAGCGCGAGTCCCGTGGTCTGTTCGGCGCTATCGCTGGTTTCATCGAGGGCGGTTGGCAGGGCATGGTGGACGGATGGTACGGTTACCACCACTCTAACGAGCAGGGTTCCGGTTACGCTGCTGACAAGGAGTCCACCCAGAAGGCTATCGACGGCGTCACCAACAAGGTGAACTCCATCATCGACAAGATGAACACCCAGTTCGAGGCTGTGGGTCGTGAGTTCAACAACCTCGAGCGTCGTATCGAGAACCTGAACAAGAAGATGGAGGACGGTTTCCTGGACGTGTGGACCTACAACGCCGAGCTGCTGGTGCTGATGGAGAACGAGCGTACCCTGGACTTCCACGACTCCAACGTGAAGAACCTGTACGACAAGGTCCGCCTGCAGCTGCGTGACAACGCTAAGGAGCTGGGTAACGGTTGCTTCGAGTTCTACCACAAGTGCGACAACGAGTGCATGGAGTCCATCCGTAACGGCACCTACAACTACCCCCAGTACTCCGAGGAGGCTCGTCTGAAGCGTGAGGAGATCTCCGGCGTGAAGCTCGAGTCCATCGGAACCTACCAGATCCTGTCCATCTACTCCACCGTGGCTTCCTCCCTGGCTCTGGCTATCATGATGGCTGGTCTGTCCCTGTGGATGTGCTCCAACGGT

A/INDONESIA HA Protein sequence  (SEQ ID 45)MEKIVLLLAI VSLVKSDQIC IGYHANNSTE QVDTIMEKNV TVTHAQDILEKTHNGKLCDL DGVKPLILRD CSVAGWLLGN PMCDEFINVP EWSYIVEKANPTNDLCYPGS FNDYEELKHL LSRINHFEKI QIIPKSSWSD HEASSGVSSACPYLGSPSFF RNVVWLIKKN STYPTIKKSY NNTNQEDLLV LWGIHHPNDAAEQTRLYQNP TTYISIGTST LNQRLVPKIA TRSKVNGQSG RMEFFWTILKPNDAINFESN GNFIAPEYAY KIVKKGDSAI MKSELEYGNC NTKCQTPMGAINSSMPFHNI HPLTIGECPK YVKSNRLVLA TGLRNSPQRE SRGLFGAIAGFIEGGWQGMV DGWYGYHHSN EQGSGYAADK ESTQKAIDGV TNKVNSIIDKMNTQFEAVGR EFNNLERRIE NLNKKMEDGF LDVWTYNAEL LVLMENERTLDFHDSNVKNL YDKVRLQLRD NAKELGNGCF EFYHKCDNEC MESIRNGTYNYPQYSEEARL KREEISGVKL ESIGTYQILS IYSTVASSLA LAIMMAGLSL WMCSNGSLQC RICIA/INDONESIA Optimized NA (Start and stop codon are underlined)(SEQ ID 46)

ATGGTGATCGGTATCGTGTCCCTGATGCTGCAGATCGGTAACATGATCTCCATCTGGGTGTCCCACTCCATCCAGACCGGTAACCAGCACCAGGCTGAGTCCATCTCCAACACCAACCCCCTGACCGAGAAGGCTGTGGCTTCCGTGACCCTGGCTGGTAACTCCTCCCTGTGCCCCATCCGTGGTTGGGCTGTGCACTCCAAGGACAACAACATCCGCATCGGTTCCAAGGGTGACGTGTTCGTGATCCGTGAGCCCTTCATCTCCTGCTCCCACCTCGAGTGCCGTACCTTCTTCCTGACCCAAGGTGCTCTGCTGAACGACAAGCACTCCAACGGCACCGTGAAGGACCGTTCCCCCCACCGTACCCTGATGTCCTGCCCCGTGGGCGAGGCTCCCTCCCCCTACAACTCCCGTTTCGAGTCCGTGGCTTGGTCCGCTTCCGCTTGCCACGACGGCACCTCTTGGCTGACCATCGGTATCTCCGGTCCCGACAACGAGGCTGTCGCTGTGCTGAAGTACAACGGCATCATCACCGACACCATCAAGTCCTGGCGTAACAACATCCTGCGTACCCAGGAGTCCGAGTGCGCTTGCGTGAACGGTTCCTGCTTCACCGTGATGACCGACGGTCCCTCCGACGGCCAGGCTTCCTACAAGATCTTCAAGATGGAGAAGGGCAAGGTGGTGAAGTCCGTGGAGCTGGACGCTCCCAACTACCACTACGAGGAGTGCTCTTGCTACCCCGACGCTGGCGAGATCACCTGCGTGTGCCGTGACAACTGGCACGGTTCCAACCGTCCCTGGGTGTCCTTCAACCAGAACCTCGAGTACCAGATCGGTTACATCTGCTCCGGCGTGTTCGGTGACAACCCCCGTCCCAACGACGGAACCGGTTCCTGCGGTCCCATGTCCCCCAACGGTGCTTACGGTGTCAAGGGCTTCTCCTTCAAGTACGGTAACGGTGTCTGGATCGGTCGTACCAAGTCCACCAACTCCCGCTCCGGTTTCGAGATGATCTGGGACCCCAACGGTTGGACCGGCACCGACTCTTCCTTCTCCGTGAAGCAGGACATCGTGGCTATCACCGACTGGTCCGGTTACTCCGGTTCCTTCGTGCAGCACCCCGAGCTGACCGGTCTGGACTGCATTCGTCCCTGCTTCTGGGTGGAGCTGATCCGTGGTCGTCCCAAGGAGTCCACCATCTGGACCTCCGGCTCCTCCATCTCTTTCTGCGGTGTGAACTCCGACACCGTGTCCTGGTCCTGGCCCGACGGTGCCGAGCTGCCCTTCACCATCGACAAGTAATGAAAGCTTGAG CTCA/INDONESIA NA Protein sequence  (SEQ ID 47)MNPNQKIITI GSICMVIGIV SLMLQIGNMI SIWVSHSIQT GNQHQAESISNTNPLTEKAV ASVTLAGNSS LCPIRGWAVH SKDNNIRIGS KGDVFVIREPFISCSHLECR TFFLTQGALL NDKHSNGTVK DRSPHRTLMS CPVGEAPSPYNSRFESVAWS ASACHDGTSW LTIGISGPDN EAVAVLKYNG IITDTIKSWRNNILRTQESE CACVNGSCFT VMTDGPSDGQ ASYKIFKMEK GKVVKSVELDAPNYHYEECS CYPDAGEITC VCRDNWHGSN RPWVSFNQNL EYQIGYICSGVFGDNPRPND GTGSCGPMSP NGAYGVKGFS FKYGNGVWIG RTKSTNSRSGFEMIWDPNGW TGTDSSFSVK QDIVAITDWS GYSGSFVQHP ELTGLDCIRPCFWVELIRGR PKESTIWTSG SSISFCGVNS DTVSWSWPDG AELPFTIDKA/INDONESIA Optimized M1  (SEQ ID 48)

ATCCCCTCCGGTCCTCTGAAGGCTGAGATCGCTCAGAAGCTCGAGGACGTTTTCGCTGGCAAGAACACCGACCTCGAGGCTCTGATGGAGTGGCTCAAGACCCGTCCCATCCTGTCCCCCCTGACCAAGGGTATCCTGGGTTTCGTGTTCACCCTGACCGTGCCCTCCGAGCGTGGTCTGCAGCGTCGTCGTTTCGTGCAGAACGCTCTGAACGGTAACGGTGACCCCAACAACATGGACCGTGCTGTGAAGCTGTACAAGAAGCTGAAGCGCGAGATCACCTTCCACGGTGCTAAGGAGGTGTCCCTGTCCTACTCCACCGGTGCTCTGGCTAGCTGCATGGGCCTGATCTACAACCGTATGGGCACCGTGACCACCGAGGTGGCCTTCGGTCTGGTCTGCGCTACCTGCGAGCAGATCGCTGACTCCCAGCACCGTTCCCACCGTCAGATGGCTACCATCACCAACCCCCTGATCCGTCACGAGAACCGTATGGTGCTGGCTTCCACCACCGCTAAGGCTATGGAGCAGATGGCTGGTTCCTCCGAGCAGGCTGCTGAGGCCATGGAGGTGGCCAACCAGGCTCGTCAGATGGTGCAGGCTATGCGTACCATCGGCACCCACCCCAACTCCTCCGCTGGTCTGCGTGACAACCTGCTCGAGAACCTGCAGGCTTACCAGAAGCGTATGGGAGTCCAGATGCAGCGCTTCAAGTAATGA

A/INDONESIA M1 Protein sequence  (SEQ ID 49)MSLLTEVETY VLSIIPSGPL KAEIAQKLED VFAGKNTDLE ALMEWLKTRPILSPLTKGIL GFVFTLTVPS ERGLQRRRFV QNALNGNGDP NNMDRAVKLYKKLKREITFH GAKEVSLSYS TGALASCMGL IYNRMGTVTT EVAFGLVCATCEQIADSQHR SHRQMATITN PLIRHENRMV LASTTAKAME QMAGSSEQAAEAMEVANQAR QMVQAMRTIG THPNSSAGLR DNLLENLQAY QKRMGVQMQR FK A/Anhui/1/2005A/Anhui Optimized HA (Start and stop codon are underlined)  (SEQ ID 50)

AAGTCCGACCAGATCTGCATCGGTTACCACGCTAACAACTCCACCGAGCAGGTGGACACCATCATGGAGAAGAACGTCACCGTGACCCACGCTCAGGACATCCTGGAAAAGACCCACAACGGCAAGCTGTGCGACCTGGACGGTGTCAAGCCCCTGATCCTGCGTGACTGCTCCGTGGCTGGTTGGCTGCTGGGTAACCCCATGTGCGACGAGTTCATCAACGTGCCCGAGTGGTCCTACATCGTGGAGAAGGCTAACCCCGCTAACGACCTGTGCTACCCCGGTAACTTCAACGACTACGAGGAGCTGAAGCACCTGCTGTCCCGTATCAACCACTTCGAGAAGATCCAGATCATCCCCAAGTCCTCTTGGTCCGACCACGAGGCTTCCTCCGGTGTCTCCTCCGCTTGCCCATACCAGGGCACCCCATCTTTCTTCCGTAACGTGGTGTGGCTGATCAAGAAGAACAACACCTACCCCACCATCAAGCGTTCCTACAACAACACCAACCAGGAGGACCTGCTGATCCTGTGGGGTATCCACCACTCCAACGACGCTGCCGAGCAGACCAAGCTGTACCAGAACCCCACCACCTACATCTCCGTGGGCACCTCCACCCTGAACCAGCGTCTGGTGCCCAAGATCGCTACCCGTTCCAAGGTGAACGGCCAGTCCGGTCGTATGGACTTCTTCTGGACCATCCTGAAGCCTAACGACGCTATCAACTTCGAGTCCAACGGCAACTTCATCGCTCCCGAGTACGCTTACAAGATCGTGAAGAAGGGCGACTCCGCTATCGTCAAGTCCGAGGTGGAGTACGGTAACTGCAACACCAAGTGCCAGACCCCCATCGGTGCTATCAACTCCTCCATGCCCTTCCACAACATCCACCCCCTGACCATCGGCGAGTGCCCCAAGTACGTGAAGTCCAACAAGCTGGTGCTGGCTACCGGTCTGCGTAACTCCCCCCTGCGTGAGCGTGGTCTGTTCGGCGCTATCGCTGGTTTCATCGAGGGCGGTTGGCAGGGCATGGTGGACGGTTGGTACGGTTACCACCACAGCAACGAGCAGGGTTCCGGTTACGCTGCTGACAAGGAGTCCACCCAGAAGGCTATCGACGGCGTCACCAACAAGGTGAACTCCATCATCGACAAGATGAACACCCAGTTCGAGGCTGTGGGTCGTGAGTTCAACAACCTGGAGCGTCGTATCGAGAACCTGAACAAGAAGATGGAGGACGGTTTCCTGGACGTGTGGACCTACAACGCCGAGCTGCTGGTGCTGATGGAGAACGAGCGTACCCTGGACTTCCACGACTCTAACGTGAAGAACCTGTACGACAAGGTCCGCCTGCAGCTGCGTGACAACGCTAAGGAGCTGGGTAACGGTTGCTTCGAGTTCTACCACAAGTGCGACAACGAGTGCATGGAGTCCGTGCGTAACGGCACCTACGACTACCCCCAGTACTCCGAGGAGGCTCGTCTGAAGCGTGAGGAGATCTCCGGCGTGAAGCTGGAGTCCATCGGCACCTACCAGATCCTGTCCATCTACTCCACCGTGGCTTCCTCCCTGGCTCTGGCTATCATGGTGGCTGGTCTGTCCCTGTGGATGTGCTCCAAC

A/Anhui HA Protein sequence (SEQ ID 51)MEKIVLLLAI VSLVKSDQIC IGYHANNSTE QVDTIMEKNV TVTHAQDILEKTHNGKLCDL DGVKPLILRD CSVAGWLLGN PMCDEFINVP EWSYIVEKANPANDLCYPGN FNDYEELKHL LSRINHFEKI QIIPKSSWSD HEASSGVSSACPYQGTPSFF RNVVWLIKKN NTYPTIKRSY NNTNQEDLLI LWGIHHSNDAAEQTKLYQNP TTYISVGTST LNQRLVPKIA TRSKVNGQSG RMDFFWTILKPNDAINFESN GNFIAPEYAY KIVKKGDSAI VKSEVEYGNC NTKCQTPIGAINSSMPFHNI HPLTIGECPK YVKSNKLVLA TGLRNSPLRE RGLFGAIAGFIEGGWQGMVD GWYGYHHSNE QGSGYAADKE STQKAIDGVT NKVNSIIDKMNTQFEAVGRE FNNLERRIEN LNKKMEDGFL DVWTYNAELL VLMENERTLDFHDSNVKNLY DKVRLQLRDN AKELGNGCFE FYHKCDNECM ESVRNGTYDYPQYSEEARLK REEISGVKLE SIGTYQILSI YSTVASSLAL AIMVAGLSLW MCSNGSLQCR ICIA/Bar headed goose/Qinghai/1A/2005A/Qinghai Optimized HA (Start and stop codon are underlined) (SEQ ID 52)CGGGCGCGGAGCGGCCGCATGGAGAAGATCGTGCTGCTGCTGGCTATCGTGTCTCTGGTCAAGTCCGACCAGATCTGCATCGGTTACCACGCTAACAACTCCACCGAGCAGGTGGACACCATCATGGAGAAGAACGTCACCGTGACCCACGCTCAGGACATCCTCGAAAAGACCCACAACGGCAAGCTGTGCGACCTGGACGGCGTGAAGCCCCTGATCCTGCGTGACTGCTCCGTGGCTGGTTGGCTGCTGGGTAACCCCATGTGCGACGAGTTCCTCAACGTGCCCGAGTGGTCCTACATCGTGGAGAAGATCAACCCCGCTAACGACCTGTGCTACCCCGGTAACTTCAACGACTACGAGGAGCTGAAGCACCTGCTGTCCCGTATCAACCACTTCGAGAAGATCCAGATCATCCCCAAGTCCTCTTGGTCCGACCACGAGGCTTCCTCCGGTGTCTCCTCCGCTTGCCCATACCAGGGCCGTTCTTCCTTCTTCCGCAACGTGGTGTGGCTGATCAAGAAGAACAACGCCTACCCCACCATCAAGCGTTCCTACAACAACACCAACCAGGAGGACCTGCTGGTCCTGTGGGGTATCCACCACCCCAACGACGCTGCCGAGCAGACCCGTCTGTACCAGAACCCCACCACCTACATCTCCGTGGGCACCTCTACCCTGAACCAGCGTCTGGTGCCCAAGATCGCTACCCGTTCCAAGGTGAACGGCCAGTCCGGTCGTATGGAGTTCTTCTGGACCATCCTGAAGCCTAACGACGCTATCAACTTCGAGTCCAACGGCAACTTCATCGCTCCCGAGAACGCTTACAAGATCGTGAAGAAGGGCGACTCCACCATCATGAAGTCCGAGCTGGAGTACGGCAACTGCAACACTAAGTGCCAGACCCCCATCGGTGCTATCAACTCCTCCATGCCCTTCCACAACATCCACCCCCTGACTATCGGCGAGTGCCCCAAGTACGTGAAGTCCAACCGTCTGGTGCTGGCTACCGGTCTGCGTAACTCCCCCCAGATCGAGACTCGTGGTCTGTTCGGCGCTATCGCTGGTTTCATCGAGGGCGGTTGGCAGGGCATGGTGGACGGTTGGTACGGTTACCACCACTCTAACGAGCAGGGTTCCGGTTACGCTGCTGACAAGGAGTCTACCCAGAAGGCTATCGACGGCGTCACCAACAAGGTGAACTCCATCATCGACAAGATGAACACCCAGTTCGAGGCTGTGGGTCGTGAGTTCAACAACCTCGAACGTCGTATCGAGAACCTGAACAAGAAGATGGAGGACGGTTTCCTGGACGTGTGGACCTACAACGCCGAGCTGCTGGTGCTGATGGAGAACGAGCGTACCCTGGACTTCCACGACTCCAACGTGAAGAACCTGTACGACAAGGTCCGCCTGCAGCTGCGTGACAACGCTAAGGAGCTGGGTAACGGTTGCTTCGAGTTCTACCACCGTTGCGACAACGAGTGCATGGAGTCCGTGCGTAACGGCACCTACGACTACCCCCAGTACTCCGAGGAGGCTCGTCTGAAGCGTGAGGAGATCTCCGGTGTCAAGCTCGAATCCATCGGAACCTACCAGATCCTGTCCATCTACTCCACCGTGGCTTCCTCCCTGGCTCTGGCTATCATGGTGGCTGGT

A/Qinghai HA Protein sequence (SEQ ID 53)MEKIVLLLAI VSLVKSDQIC IGYHANNSTE QVDTIMEKNV TVTHAQDILEKTHNGKLCDL DGVKPLILRD CSVAGWLLGN PMCDEFLNVP EWSYIVEKINPANDLCYPGN FNDYEELKHL LSRINHFEKI QIIPKSSWSD HEASSGVSSACPYQGRSSFF RNVVWLIKKN NAYPTIKRSY NNTNQEDLLV LWGIHHPNDAAEQTRLYQNP TTYISVGTST LNQRLVPKIA TRSKVNGQSG RMEFFWTILKPNDAINFESN GNFIAPENAY KIVKKGDSTI MKSELEYGNC NTKCQTPIGAINSSMPFHNI HPLTIGECPK YVKSNRLVLA TGLRNSPQIE TRGLFGAIAGFIEGGWQGMV DGWYGYHHSN EQGSGYAADK ESTQKAIDGV TNKVNSIIDKMNTQFEAVGR EFNNLERRIE NLNKKMEDGF LDVWTYNAEL LVLMENERTLDFHDSNVKNL YDKVRLQLRD NAKELGNGCF EFYHRCDNEC MESVRNGTYDYPQYSEEARL KREEISGVKL ESIGTYQILS IYSTVASSLA LAIMVAGLSL WMCSNGSLQC RICIA/Qinghai Optimized NA (Start and stop codon are underlined) (SEQ ID 54)

GATCGGTATCGTGTCCCTGATGCTGCAGATCGGTAACATGATCTCCATCTGGGTGTCCCACTCCATCCAGACCGGTAACCAGCGTCAGGCCGAGCCCATCTCCAACACCAAGTTCCTCACCGAGAAGGCTGTGGCTTCCGTGACCCTGGCTGGTAACTCCTCCCTGTGCCCCATCTCCGGTTGGGCTGTGTACTCCAAGGACAACTCCATCCGTATCGGTTCCCGTGGTGACGTGTTCGTGATCCGTGAGCCCTTCATCTCCTGCTCCCACCTCGAATGCCGTACCTTCTTCCTGACCCAGGGTGCTCTGCTGAACGACAAGCACTCCAACGGCACCGTGAAGGACCGTTCCCCCCACCGTACCCTGATGTCCTGCCCCGTGGGCGAGGCTCCCTCCCCCTACAACTCCCGTTTCGAGTCCGTGGCTTGGTCCGCTTCCGCTTGCCACGACGGCACCTCTTGGCTGACCATCGGTATCTCCGGTCCCGACAACGGTGCTGTGGCTGTGCTGAAGTACAACGGCATCATCACCGACACCATCAAGTCCTGGCGTAACAACATCCTGCGTACCCAAGAGTCCGAGTGCGCTTGCGTGAACGGTTCCTGCTTCACCGTGATGACCGACGGTCCCTCCAACGGCCAGGCTTCCTACAAGATCTTCAAGATGGAGAAGGGCAAGGTGGTGAAGTCCGTGGAGCTGGACGCTCCCAACTACCACTACGAGGAGTGCTCTTGCTACCCCGACGCTGGCGAGATCACCTGCGTGTGCCGTGACAACTGGCACGGTTCCAACCGTCCCTGGGTGTCCTTCAACCAGAACCTCGAATACCAGATCGGTTACATCTGCTCCGGCGTGTTCGGTGACAACCCCCGTCCCAACGACGGAACCGGTTCCTGCGGTCCCGTGTCCCCCAACGGTGCTTACGGTGTCAAGGGCTTCTCCTTCAAGTACGGTAACGGTGTCTGGATCGGTCGTACCAAGTCCACCAACTCCCGCTCCGGTTTCGAGATGATCTGGGACCCCAACGGTTGGACCGGCACCGACTCTTCCTTCTCCGTGAAGCAGGACATCGTGGCTATCACCGACTGGTCCGGTTACTCCGGTTCCTTCGTGCAGCACCCCGAGCTGACCGGTCTGGACTGTATCCGTCCCTGCTTCTGGGTGGAGCTGATCCGTGGTCGTCCCAAGGAGTCCACCATCTGGACCTCCGGCTCCTCCATCTCTTTCTGCGGTGTGAACTCCGACACCGTGTCCTGGTCCTGG

Protein sequence: A/Qinghai NA Protein sequence (SEQ ID 55)MNPNQKIITI GSICMVIGIV SLMLQIGNMI SIWVSHSIQT GNQRQAEPISNTKFLTEKAV ASVTLAGNSS LCPISGWAVY SKDNSIRIGS RGDVFVIREPFISCSHLECR TFFLTQGALL NDKHSNGTVK DRSPHRTLMS CPVGEAPSPYNSRFESVAWS ASACHDGTSW LTIGISGPDN GAVAVLKYNG IITDTIKSWRNNILRTQESE CACVNGSCFT VMTDGPSNGQ ASYKIFKMEK GKVVKSVELDAPNYHYEECS CYPDAGEITC VCRDNWHGSN RPWVSFNQNL EYQIGYICSGVFGDNPRPND GTGSCGPVSP NGAYGVKGFS FKYGNGVWIG RTKSTNSRSGFEMIWDPNGW TGTDSSFSVK QDIVAITDWS GYSGSFVQHP ELTGLDCIRPCFWVELIRGR PKESTIWTSG SSISFCGVNS DTVSWSWPDG AELPFTIDKThe following references are incorporated herein by reference:

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OTHER EMBODIMENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims:

The invention claimed is:
 1. A vaccine comprising an influenza VLP,wherein said VLP comprises influenza M1, HA and NA proteins, whereinsaid vaccine induces substantial immunity to influenza virus infectionin an animal susceptible to influenza, wherein the M1 protein is derivedfrom A/Indonesia/5/05, wherein the M1 protein is derived from adifferent influenza virus strain as compared to the HA and NA proteins.2. The vaccine of claim 1, wherein said HA and/or NA exhibithemagglutinin activity and/or neuraminidase activity, respectfully. 3.The vaccine of claim 1, wherein said influenza VLP comprises seasonalinfluenza virus HA and NA proteins.
 4. The vaccine of claim 3, whereinsaid seasonal influenza virus is a type A influenza virus.
 5. Thevaccine of claim 3, wherein said seasonal influenza virus is a type Binfluenza virus.
 6. The vaccine of claim 1, wherein said influenza VLPcomprises avian influenza HA and NA proteins.
 7. The vaccine of claim 6,wherein said avian influenza virus HA and NA proteins are H5N1.
 8. Thevaccine of claim 6, wherein said avian influenza virus HA and NAproteins are H9N2.
 9. The vaccine of claim 1, wherein said animal is ahuman.
 10. The vaccine of claim 1, wherein said influenza VLP isformulated with an adjuvant or immune stimulator.
 11. The vaccine ofclaim 10, wherein said adjuvant comprises paucilamellar non-phospholipidvesicles.
 12. The vaccine of claim 1, wherein said vaccine comprises atleast a second VLP which comprises HA and NA from different influenzastrains.
 13. The vaccine of claim 1, wherein said VLP consistsessentially of influenza M1, HA and NA proteins.
 14. The vaccine ofclaim 1, wherein said VLP consists of influenza M1, HA and NA proteins.15. A method of formulating a vaccine that induces substantial immunityto influenza virus infection to an animal susceptible to influenza,comprising adding an effective dose of an influenza VLP to apharmaceutically acceptable carrier or excipient, wherein said VLPcomprises influenza M1, HA and NA proteins, wherein said vaccine inducessubstantial immunity to influenza virus infection to said animal,wherein the M1 protein is derived from A/Indonesia/5/05, wherein the M1protein is derived from a different influenza virus strain as comparedto the HA and NA proteins.
 16. The method of claim 15, wherein said VLPconsists essentially of influenza M1, HA and NA proteins.
 17. The methodof claim 15, wherein said VLP consists of influenza M1, HA and NAproteins.
 18. The method of claim 15, wherein said influenza VLP hasbeen treated to inactivate baculovirus.
 19. The method of claim 18,wherein said inactivation treatment comprises incubating a samplecomprising VLPs in about 0.2% of β-propyl lactone (BPL) for about 3hours at about 25° C.
 20. A virus like particle (VLP) comprising aninfluenza virus M1 protein and influenza virus HA and NA hemagglutininand neuraminidase proteins, wherein the M1 protein is derived fromA/Indonesia/5/05, wherein the M1 protein is derived from a differentinfluenza virus strain as compared to the HA and NA proteins.
 21. TheVLP of claim 20, wherein said HA or NA are from a H5N1 clade 1 influenzavirus.
 22. The VLP of claim 20, wherein said HA and NA are from a H5N1clade 2 influenza virus.
 23. The VLP of claim 22, wherein said HA and NAproteins comprise SEQ ID NOS: 50 and 54, respectively.
 24. The VLP ofclaim 20, wherein said HA and NA are from an influenza virus which wasisolated from an infected animal.
 25. The VLP of claim 24, wherein saidinfected animal is a human.
 26. The VLP of claim 20, wherein the VLP isexpressed from a eukaryotic cell comprising one or more nucleic acidsencoding influenza HA and NA proteins and an influenza M1 protein underconditions that permit the formation of VLPs.
 27. The VLP of claim 26,wherein said eukaryotic cell is selected from the group consisting ofyeast, insect, amphibian, avian and mammalian cells.
 28. The VLP ofclaim 27, wherein said eukaryotic cell is an insect cell.
 29. The VLP ofclaim 28, wherein said insect cell is Sf9.
 30. The VLP of claim 20,wherein said VLP elicits neutralizing antibodies in a human or animalthat are protective against influenza infection when administered tosaid human or animal.
 31. An immunogenic composition comprising aneffective dose of a VLP of claim
 20. 32. The composition of claim 31,wherein said composition comprises an adjuvant.
 33. A vaccine comprisingan effective dose of a VLP of claim
 20. 34. The vaccine of claim 33,wherein said vaccine comprises at least a second VLP which comprises HAand NA from different influenza strains.
 35. The vaccine of claim 33,wherein said vaccine comprises an adjuvant.
 36. The vaccine of claim 1wherein the M1 protein derived from A/Indonesia/5/05 comprises SEQ IDNO:
 49. 37. The method of claim 15 wherein the M1 protein derived fromA/Indonesia/5/05 comprises SEQ ID NO:
 49. 38. The VLP of claim 20wherein the M1 protein derived from A/Indonesia/5/05 comprises SEQ IDNO: 49.